Polypeptide having α-isomaltosyl-transferase activity

ABSTRACT

The object of the present invention is to provide a polypeptide which can be used to produce a saccharide having a structure of cyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→}, a DNA encoding the polypeptide, and uses thereof. The present invention solves the above object by establishing a polypeptide which has an enzymatic activity to produce a saccharide having a structure of cyclo{→6}-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→} from a saccharide with a glucose polymerization degree of 3 or higher and bearing both the α-1,6 glucosidic linkage as a linkage at the non-reducing end and the α-1,4 glucosidic linkage other than the linkage at the non-reducing end by catalyzing the α-isomaltosyl-transfer, and having an amino acid sequence of either SEQ ID NO:1 or SEQ ID NO:2, or that which is a member selected from the group consisting of amino acid sequences having deletion, replacement, or addition of one or more amino acid residues therein or thereto, a DNA encoding the polypeptide, and uses thereof.

FIELD OF THE INVENTION

The present invention relates to polypeptides which forming a cyclictetrasaccharide having the structure of cyclo{ 6)-α-D-glucopyranosyl-(13)-α-D-glucopyranosyl-(1 6)-α-D-glucopyranosyl-(13)-α-D-glucopyranosyl-(1 } from a saccharide with a glucosepolymerization degree of 3 or higher and bearing both the α-1,6glucosidic linkage as a linkage at the non-reducing end and the α-1,4glucosidic linkage other than the linkage at the non-reducing end, andwhich comprise an amino acid sequence of either SEQ ID NO:1 or SEQ IDNO:2, or the amino acid sequence having deletion, replacement, oraddition of one or more amino acid residues of SEQ ID NO:1 or SEQ IDNO:2; and to uses thereof. More particularly, the present inventionrelates to polypeptides which form a cyclic tetrasaccharide having astructure of cyclo{6)-α-D-glucopyranosyl-(1 3)-α-D-glucopyranosyl-(16)-α-D-glucopyranosyl-(1 3)-α-D-glucopyranosyl-(1} from a saccharidewith a glucose polymerization degree of 3 or higher and bearing both theα-1,6 glucosidic linkage as a linkage at the non-reducing end and theα-1,4 glucosidic linkage other than the linkage at the non-reducing end,and which comprise the amino acid sequence of either SEQ ID NO:1 or SEQID NO:2, or the amino acid sequence having deletion, replacement oraddition of one or more amino acid residues of SEQ ID NO:1 or SEQ IDNO:2, to a DNA encoding the amino acid sequence, to a replicablerecombinant DNA which comprises a DNA encoding the polypeptide and anautonomously replicable vector, to transformants which are constructedby introducing the recombinant DNAs into appropriate hosts, to a processfor preparing the polypeptides, to the cyclic tetrasaccharide describedabove, and to uses thereof.

BACKGROUND ART

There have been known several saccharides which are composed of glucosemolecules as constituents, for example, partial starch hydrolyzates,produced from starches as materials, including amyloses, amylodextrins,maltodextrins, maltooligosaccharides, and isomaltooligosaccharides.Also, these saccharides are known to have usually non-reducing andreducing groups at their molecular ends and exhibit reducibility.Usually, partial starch hydrolyzates, which have a strong reducing poweron a dry solid basis, are known to have properties of a relatively lowmolecular weight and viscosity, a relatively strong sweetness andreactivity, easy reactivity with amino group-containing substances suchas amino acids and proteins by amino carbonyl reaction that may inducebrowning and unpleasant smell, and easily cause deterioration.Therefore, methods for decreasing or eliminating the reducing power ofreducing saccharides without altering glucose residues have beenrequired for a long time. For example, as disclosed in “Journal ofAmerican Chemical Society, Vol. 71, 353–358 (1949)”, it was reportedthat methods for forming α-, β- or γ-cyclodextrins that are composed of6, 7 or 8 glucose molecules linked together via the α-1,4 glucosidiclinkage by contacting “macerans amylase” with starches. Nowadays, thesecyclodextrins are produced on an industrial scale and are used indiversified fields using their inherent properties such asnon-reducibility, tasteless, and inclusion abilities. As disclosed, forexample, in Japanese Patent Kokai Nos. 143,876/95 and 213,283 appliedfor by the same applicant of the present invention, it is known a methodfor producing trehalose, composed of two glucose molecules linkedtogether via the α,α-linkage, by contacting a non-reducingsaccharide-forming enzyme and a trehalose-releasing enzyme with partialstarch hydrolyzates such as maltooligosaccharides. At present, trehalosehas been industrially produced from starches and used in differentfields by using its advantageous non-reducibility, mild- and highquality-sweetness. As described above, trehalose having a glucosepolymerization degree of 2, and α-, β- and γ-cyclodextrin having aglucose polymerization degree of 6, 7 and 8, are produced on anindustrial scale and used in view of their advantageous properties,however, the types of non- or low-reducing saccharides are limited, sothat more diversified saccharides other than these saccharide aregreatly required.

Recently, a novel cyclic tetrasaccharide constructed by glucoses hasbeen disclosed. For example, “European Journal of Biochemistry, Vol.226, 641–648 (1994)” shows that a cyclic tetrasaccharide which has astructure ofcyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→}(hereinafter, called “cyclotetrasaccharide” in the presentspecification, unless specified otherwise.) is formed by contacting ahydrolyzing enzyme, alternanase, with alternan linked with glucosemolecules via the alternating α-1,3 and α-1,6 bonds, followed bycrystallization under the co-existence of methanol. Sincecyclotetrasaccharide is a sugar having a cyclic structure and has noreducing power, it is expected that the saccharide shows noamino-carbonyl reactivity, and is useful to stabilize volatile organiccompounds by its inclusion ability, and to be processed without anyapprehension of browning and deterioration. However, it has beendifficult to obtain alternan as a material and alternanase as an enzymefor preparing cyclotetrasaccharide. In addition, it has beensubstantially difficult to obtain a microorganism producing the enzyme.

Under these circumstances, the present inventors made every effort tostudy on a novel process for industrial production ofcyclotetrasaccharide. As disclosed in PTC/JP01/04276, the presentinventors found microorganisms of the genera Bacillus and Arthrobacterwhich produce an absolutely novel and ever unknown enzyme,α-isomaltosyl-transferring enzyme for forming cyclotetrasaccharide froma saccharide with a glucose polymerization degree of 3 or higher andbearing both the α-1,6 glucosidic linkage as a linkage at thenon-reducing end and the α-1,4 glucosidic linkage other than the linkageat the non-reducing end. They found and disclosed in PCT/JP01/06412 thatthese microorganisms also produced another novel enzyme,α-isomaltosylglucosaccharide-forming enzyme which forms a saccharidewith a glucose polymerization degree of 3 or higher and bearing both theα-1,6 glucosidic linkage as a linkage at the non-reducing end and theα-1,4 glucosidic linkage other than the linkage at the non-reducing endfrom saccharides with a glucose polymerization degree of 2 or higher.Furthermore, the present inventors found that cyclotetrasaccharide canbe obtained from starchy saccharides with a glucose polymerizationdegree of 3 or higher by using α-isomaltosyl-transferring enzyme andα-isomaltosylglucosaccharide-forming enzyme. However, since theproductivities of α-isomaltosyl-transferring enzyme of thesemicroorganisms were not enough, a large-scale cultivation of thesemicroorganisms is substantially difficult for industrial scaleproduction of cyclotetrasaccharide.

Now, it has been revealed that the entity of the enzyme is apolypeptide, and the enzymatic activity is controlled by its amino acidsequence, as well as a DNA that encodes the amino acid sequence.Therefore, if a gene which encodes the polypeptide will be isolated, andif its nucleotide sequence will be determined, it will be relativelyeasy to prepare the desired amount of the polypeptide by a method whichcomprises the steps of constructing a recombinant DNA containing a genewhich encodes the polypeptide, introducing the recombinant DNA intohost-cells of microorganisms, animals or plants, and culturing theobtained transformants.

Under these circumstances, required are the isolation of a gene encodinga polypeptide as the entity of α-isomaltosyl-transferring enzyme,sequencing of the nucleotide sequence, and stable preparation of thepolypeptide in large scale and at a relatively low cost.

DISCLOSURE OF INVENTION

The first object of the present invention is to establish a polypeptidewhich has α-isomaltosyl-transferring enzymatic activity which catalyzesthe formation of cyclotetrasaccharide from a saccharide with a glucosepolymerization degree of 3 or higher and bearing both the α-1,6glucosidic linkage as a linkage at the non-reducing end and the α-1,4glucosidic linkage other than the linkage at the non-reducing end(hereinafter, the polypeptide described above may be abbreviated as “thepolypeptide of the present invention”).

The second object of the present invention is to provide a DNA encodingthe polypeptide of the present invention.

The third object of the present invention is to provide a replicablerecombinant DNA comprising the DNA.

The fourth object of the present invention is to provide a transformanttransformed by the recombinant DNA.

The fifth object of the present invention is to provide a process forproducing the polypeptide of the present invention by using thetransformant.

The sixth object of the present invention is to provide a process forforming cyclotetrasaccharide from a saccharide with a glucosepolymerization degree of 3 or higher and bearing both the α-1,6glucosidic linkage as a linkage at the non-reducing end and the α-1,4glucosidic linkage other than the linkage at the non-reducing end byusing the polypeptide of the present invention.

The seventh object of the present invention is to providecyclotetrasaccharide, which can be obtained using the polypeptide of thepresent invention, and to its uses.

The present invention solves the first object by providing a polypeptidewhich forms cyclotetrasaccharide having a structure ofcyclo{→6}-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→)from a saccharide with a glucose polymerization degree of 3 or higherand bearing both the α-1,6 glucosidic linkage as a linkage at thenon-reducing end and the α-1,4 glucosidic linkage other than the linkageat the non-reducing end, and a polypeptide comprising the amino acidsequence of either SEQ ID NO:1 or SEQ ID NO:2, or the amino acidsequence having deletion, replacement or insertion of one or more aminoacids of SEQ ID NO:1 or SEQ ID NO:2

The present invention solves the second object described above byproviding a DNA encoding the polypeptide.

The present invention solves the third object described above byproviding a replicable recombinant DNA which comprises a DNA encodingthe polypeptide and an autonomously replicable vector.

The present invention solves the fourth object described above byproviding a transformant constructed by introducing the recombinant DNAinto an appropriate host.

The present invention solves the fifth object described above byproviding a process for preparing the polypeptide, which comprises thesteps of culturing the transformant constructed by introducing areplicable recombinant DNA, which contains a DNA encoding thepolypeptide and an autonomously replicable vector, into appropriatehosts, and collecting the polypeptide from the resultant culture.

The present invention solves the sixth object described above byproviding a process for producing cyclotetrasaccharide, which comprisesa step of forming cyclotetrasaccharide from a saccharide with a glucosepolymerization degree of 3 or higher and bearing both the α-1,6glucosidic linkage as a linkage at the non-reducing end and the α-1,4glucosidic linkage other than the linkage at the non-reducing end usingthe polypeptide of the present invention.

The present invention solves the seventh object described above byproducing cyclotetrasaccharide which is obtained by using thepolypeptide of the present invention, and providing foods, cosmetics andpharmaceuticals which comprise cyclotetrasaccharide or saccharidecompositions containing cyclotetrasaccharide.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the optimum temperature of a polypeptide havingα-isomaltosyl-transferring enzyme activity from a microorganism of thespecies Bacillus globisporus C11 strain.

FIG. 2 shows the optimum pH of a polypeptide havingα-isomaltosyl-transferring enzyme activity from a microorganism of thespecies Bacillus globisporus C11 strain.

FIG. 3 shows the thermal stability of a polypeptide havingα-isomaltosyl-transferring enzyme activity from a microorganism of thespecies Bacillus globisporus C11 strain.

FIG. 4 shows the pH stability of a polypeptide havingα-isomaltosyl-transferring enzyme activity from a microorganism of thespecies Bacillus globisporus C11 strain.

FIG. 5 shows the optimum temperature of a polypeptide havingα-isomaltosyl-transferring enzyme activity from a microorganism of thespecies Bacillus globisporus N75 strain.

FIG. 6 shows the optimum pH of a polypeptide havingα-isomaltosyl-transferring enzyme activity from a microorganism of thespecies Bacillus globisporus N75 strain.

FIG. 7 shows the thermal stability of a polypeptide havingα-isomaltosyl-transferring enzyme activity from a microorganism of thespecies Bacillus globisporus N75 strain.

FIG. 8 shows the pH stability of a polypeptide havingα-isomaltosyl-transferring enzyme activity from a microorganism of thespecies Bacillus globisporus N75 strain.

FIG. 9 shows the restriction enzyme map of a recombinant DNA, pBGC1, ofthe present invention. In the figure, a section indicated with blackbold line is a DNA encoding a polypeptide havingα-isomaltosyl-transferring enzyme activity from a microorganism of thespecies Bacillus globisporus C11 strain.

FIG. 10 shows the restriction enzyme map of a recombinant DNA, pBGC1, ofthe present invention. In the figure, a section indicated with blackbold line is a DNA encoding a polypeptide havingα-isomaltosyl-transferring enzyme activity from a microorganism of thespecies Bacillus globisporus N75 strain.

BEST MODE FOR CARRYING OUT OF THE INVENTION

The present invention was made based on the finding of absolutely noveland ever unknown enzymes which catalyze the formation ofcyclotetrasaccharide from a saccharide with a glucose polymerizationdegree of 3 or higher and bearing both the α-1,6 glucosidic linkage as alinkage at the non-reducing end and the α-1,4 glucosidic linkage otherthan the linkage at the non-reducing end. These enzymes can be obtainedas polypeptides from the culture of novel microorganisms, strain C11 andstrain N75, isolated from soils by the present inventors. The presentinventors named the strain C11 “Bacillus globisporus C11”, and depositedit on Apr. 25, 2000, in International Patent Organism DepositaryNational Institute of Advanced Industrial Science and Technology TsukubaCentral 6, 1-1, Higashi 1-Chome Tsukuba-shi, Ibaraki-ken, 305-8566,Japan. The deposition of the microorganism was accepted under theaccession number of FERM BP-7144. The present inventors also named thestrain N75 “Bacillus globisporus N75”, and deposited it on May 16, 2001,in International Patent Organism Depositary National Institute ofAdvanced Industrial Science and Technology Tsukuba Central 6, 1-1,Higashi 1-Chome Tsukuba-shi, Ibaraki-ken, 305-8566, Japan. Thedeposition of the microorganism was accepted under the accession numberof FERM BP-7591. As disclosed by the present inventors inPTC/JP01/06412, the strains C11 and N75 also produceα-isomalosylglucosaccharide-forming enzyme which form a saccharide witha glucose polymerization degree of 3 or higher and bearing both theα-1,6 glucosidic linkage as a linkage at the non-reducing end and theα-1,4 glucosidic linkage other than the linkage at the non-reducing endfrom maltodextrin with a glucose polymerization degree of 2 or higher.

The following are the bacteriological properties of the strains C11 andN75.

<Bacillus globisporus C11>

<A. Morphology>

-   -   Characteristic of cells when incubated at 27 ° C. of nutrient        broth agar;    -   Existing usually in a rod shape of 0.5–1.0×1.5–5 μm,    -   Exhibiting no polymorphism,    -   Possessing motility,    -   Forming spherical spores at an intracellular end    -   And swelled sporangia, and    -   Gram stain, positive;        <B. Cultural Property>    -   (1) Characteristics of colonies formed when incubated at 27° C.        in nutrient broth agar plate;        -   Shape: Circular colony having a diameter of 1–2 mm after 2            days incubation        -   Rim: Entire        -   Projection: Hemispherical shape        -   Gloss: Dull        -   Surface: Smooth        -   Color: Opaque and pale yellow    -   (2) Characteristics of colony formed when incubated at 27° C. in        nutrient broth agar slant;        -   Growth: Roughly medium        -   Shape: Radiative    -   (3) Characteristics of colony formed when stub cultured at        27° C. in nutrient broth agar plate;        -   Liquefying the agar plate.            <C. Physiological Properties>    -   (1) VP-test: Negative    -   (2) Indole formation: Negative    -   (3) Gas formation from nitric acid: Positive    -   (4) Hydrolysis of starch: Positive    -   (5) Formation of pigment: Forming no soluble pigment    -   (6) Urease: Positive    -   (7) Oxidase: Positive    -   (8) Catalase: Positive    -   (9) Growth conditions: Growing at a pH of 5.5–9.0 and a        temperature of 10–35° C.    -   (10) Oxygen requirement: Aerobic    -   (11) Utilization of carbon source and acid formation

Carbon source Utilization Acid formation D-Glucose + + Glycerol + +Sucrose + + Lactose + + Note: The symbol “+” means yes or positive.

-   -   (14) Mol% of guanine (G) plus cytosine (C) of DNA: 39%        <Bacillus globisporus N75>        <A. Morphology>    -   (1) Characteristic of cells when incubated at 27° C. of nutrient        broth agar;        -   Existing usually in a rod shape of 0.5–1.0×1.5–5 μm,        -   Exhibiting no polymorphism,        -   Possessing motility,        -   Forming spherical spores at an intracellular end        -   And swelled sporangia, and        -   Gram stain, positive;            <B. Cultural Property>    -   (1) Characteristics of colonies formed when incubated at 27° C.        in nutrient broth agar plate;        -   Shape: Circular colony having a diameter of 1–2 mm after 2            days incubation        -   Rim: Entire        -   Projection: Hemispherical shape        -   Gloss: Dull        -   Surface: Smooth        -   Color: Opaque and pale yellow    -   (2) Characteristics of colony formed when incubated at 27° C. in        nutrient broth agar slant;        -   Growth: Roughly medium        -   Shape: Radiative    -   (3) Characteristics of colony formed when stub cultured at        27° C. in nutrient broth agar plate;        -   Liquefying the agar plate.            <C. Physiological Properties>    -   (1) VP-test: Negative    -   (2) Indole formation: Negative    -   (3) Gas formation from nitric acid: Positive    -   (4) Hydrolysis of starch: Positive    -   (5) Formation of pigment: Forming no soluble pigment    -   (6) Urease: Positive    -   (7) Oxidase: Positive    -   (8) Catalase: Positive    -   (9) Growth conditions: Growing at a pH of 5.5–9.0 and a        temperature of 10–35° C.    -   (10) Oxygen requirement: Aerobic    -   (11) Utilization of carbon source and acid formation

Carbon source Utilization Acid formation D-Glucose + + Glycerol + +Sucrose + + Lactose + + Note: The symbol “+” means yes or positive.

-   -   (12) Mol% of guanine (G) plus cytosine (C) of DNA: 40%

The present inventors purified and characterized theα-isomaltosyl-transferring enzyme which is obtainable from the cultureof Bacillus globisporus C11 (FERM BP-7144) or Bacillus globisporus N75(FERM BP-7591). As a result, it was revealed that the enzyme has anactivity to form cyclotetrasaccharide having a structure ofcyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→}from a saccharide with a glucose polymerization degree of 3 or higherand bearing both the α-1,6 glucosidic linkage as a linkage at thenon-reducing end and the α-1,4 glucosidic linkage other than the linkageat the non-reducing end, and is polypeptide comprising the amino acidsequences of SEQ ID NO:1 or SEQ ID NO:2. In addition, thephysicochemical properties of the polypeptides are as follows;

-   -   (1) Molecular weight        -   Having a molecular weight of about 82,000 to about 132,000            daltons when determined on SDS-PAGE;    -   (2) Optimum temperature        -   Having an optimum temperature of about 50° C. when incubated            at a pH of 6.0 for 30 min;    -   (3) Optimum pH        -   Having an optimum pH of about 5.5 to 6.0 when incubated at            35° C. for 30 min;    -   (4) Thermal stability        -   Having a thermostable region at temperatures of about 45° C.            or lower when incubated at a pH of 6.0 for 60 min;    -   (5) pH Stability        -   Having a stable pH region at about 4.5 to 10.0 when            incubated at 4° C. for 24 hours;

The following experiments explain the physicochemical properties of thepolypeptide having an α-isomaltosyl-transferring enzymatic activity ofthe present invention.

Experiment 1

Preparation of a Polypeptide from Bacillus globisporus

Experiment 1-1

Preparation of Crude Polypeptide

A liquid culture medium consisting 4% (w/v) of “PINE-DEX #4”, a partialstarchhydrolyzate, 1.8% (w/v) of “ASAHIMEAST”, a yeast extract, 0.1%(w/v) of dipotassium phosphate, 0.06% (w/v) of sodium phosphate dodeca-hydrate, 0.05% (w/v) of magnesium sulfate hepta-hydrate, and water wasplaced in 500-ml Erlenmeyer flasks in a respective amount of 100 ml,sterilized by autoclaving at 121° C. for 20 min, cooled and seeded withBacillus globisporus C11 strain, FERM BP-7144, followed by culturingunder rotary-shaking conditions at 27° C. and 230 rpm for 48 hours for aseed culture.

About 20 L of a fresh preparation of the same liquid culture medium asused in the above seed culture were placed in a 30 L fermentor,sterilized by heating, and then cooled to 27° C. and inoculated with 1%(v/v) of the seed culture, followed by culturing at 27° C. and pH 6.0 to8.0 for 48 hours under aeration-agitation conditions. After thecompletion of the culture, about 1.8 units/ml ofα-isomaltosyl-transferring enzyme and about 0.55 unit/ml ofα-isomaltosylglucosaccharide-forming enzyme were detected in theresulting culture by measuring enzyme activities. About 18 L ofsupernatant obtained by centrifugation at 10,000 rpm for 30 min hadabout 1.7units/ml of α-isomaltosyl-transferring enzyme activity, i.e., atotal activity of about 30,400 units; and 0.51 unit ofα-isomaltosylglucosaccharide-forming enzyme activity, i.e., a totalenzymatic activity of about 9,180 units. It was revealed that theseenzymes were secretion polypeptides secreted in the culture.

The activity of α-isomaltosyl-transferring enzyme was measured by thefollowing assay: A substrate solution was prepared by dissolving panosein 100 mM acetate buffer (pH 6.0) to give a concentration of 2% (w/v). Areaction mixture was prepared by mixing 0.5 ml of the substrate solutionand 0.5 ml of an enzyme solution, and incubated at 35° C. for 30 min.After stopping the reaction by boiling for 10 min, the amount of glucoseformed in the reaction mixture was determined by the glucoseoxidase-peroxidase method. One unit of α-isomaltosyl-transferringactivity was defined as the amount of the enzyme that forms one μmole ofglucose per minute under the above conditions.

The Activity of α-isomaltosylglucosaccharide-forming enzyme was measuredby the following assay: A substrate solution was prepared by dissolvingmaltotriose in 100 mM acetate buffer (pH 6.0) to give a concentration of2% (w/v). A reaction mixture was prepared by mixing 0.5 ml of thesubstrate solution and 0.5 ml of an enzyme solution, and incubated at35° C. for 60 min. After stopping the reaction by boiling for 10 min,the amount of glucose formed in the reaction mixture was determined byhigh-performance liquid chromatography (HPLC). One unit ofα-isomaltosylglucosaccharide-forming activity was defined as the amountof the enzyme that forms one μmole of maltose per minute under the aboveconditions. HPLC was carried out using“SHODEX KS-801 column”, ShowaDenko K.K., Tokyo, Japan, at a column temperature of 60° C. and a flowrate of 0.5 ml/min of water, and using “RI-8012”, a differentialrefractometer commercialized by Tosho Corporation, Tokyo, Japan.

About 18 L of the culture supernatant described above were salted outwith 80% saturated ammonium sulfate solution and allowed to stand at 4°C. for 24 hours, and the formed precipitates were collected bycentrifugation at 10,000 rpm for 30 min, dissolved in 10 mM sodiumphosphate buffer (pH 7.5), and dialyzed against the same buffer toobtain about 416 ml of a crude enzyme solution. The crude enzymesolution had about 28,000 units of α-isomaltosyl-transferring enzyme and8,440 units of α-isomaltosylglucosaccharide-forming enzyme. The crudeenzyme solution was subjected to ion-exchange column chromatographyusing “SEPABEADS FP-DA13” gel, an ion-exchange resin commercialized byMitsubishi Chemical Industries, Ltd., Tokyo, Japan. Bothα-isomaltosyl-transferring enzyme andα-isomaltosylglucosaccharide-forming enzyme were eluted as non-adsorbedfractions without adsorbing on “SEPABEADS FP-DA13” gel. The non-adsorbedfraction was collected and dialyzed against 10 mM sodium phosphatebuffer (pH 7.0) with 1 M ammonium sulfate. The dialyzed solution wascentrifuged to remove impurities, and subjected to affinity columnchromatography using 500 ml of “SEPHACRYL HR S-200” gel, a gelcommercialized by Amersham Corp., Div. Amersham International, Arlingtonheights, Ill., USA. Enzymatically active components adsorbed on“SEPHACRY HR S-200” gel and, when sequentially eluted with a lineargradient decreasing from 1 M to 0 M of ammonium sulfate and a lineargradient increasing from 0 mM to 100 mM of maltotetraose, theα-isomaltosyl-transferring enzyme and theα-isomaltosylglucosaccharide-forming enzyme were separately eluted,i.e., the former was eluted with a linear gradient of ammonium sulfateat about 0 M and the latter was eluted with a linear gradient ofmaltotetraose at about 30 mM. Thus, fractions with theα-isomaltosyl-transferring enzyme activity and those with theα-isomaltosylglucosaccharide-forming enzyme activity were separatelycollected as crude polypeptides of α-isomaltosyl-transferring enzyme andα-isomaltosylglucosaccharide-forming enzyme. Further, the polypeptideshaving an α-isomaltosyl-transferring enzyme activity orα-isomaltosylglucosaccharide-forming enzyme activity were respectivelypurified and prepared by the methods described in the below.

Experiment 1-2

Purification of a Polypeptide having an α-Isomaltosyl-TransferringEnzyme Acticity

The crude polypeptide having an α-Isomaltosyl-transferring enzymeactivity obtained in Experiment1-1 was dialyzed against 10 mM sodiumphosphate buffer (pH 7.0) with 1 M ammonium sulfate. The dialyzedsolution was centrifuged to remove impurities, and subjected tohydrophobic column chromatography using 350 ml of “BUTYL-TOYOPEARL 650M”gel, a hydrophobic gel commercialized by Tosho Corporation, Tokyo,Japan. The enzyme adsorbed on “BUTYL-TOYOPEARL 650M” gel and, wheneluted with a linear gradient decreasing from 1 M to 0M of ammoniumsulfate, the enzymatically active fractions were eluted with a lineargradient of ammonium sulfate at about 0.3 M, and fractions with theenzyme activity was collected. The collected solution was dialyzedagainst 10 mM sodium phosphate buffer (pH 7.0) with 1 M ammoniumsulfate, and the dialyzed solution was centrifuged to remove impurities,and purified by affinity chromatography using “SEPHACRYL HR S-200” gel.The amount of enzyme activity, specific activity and yield of theα-isomaltosyl-transferring enzyme in each purification step are in Table1.

TABLE 1 Enzyme* Specific activity activity of enzyme* Yield Purificationstep (unit) (unit/mg protein) (%) Culture supernatant 30,400 0.45 100Dialyzed solution after 28,000 1.98 92.1 salting out with ammoniumsulfate Elute from ion-exchange 21,800 3.56 71.7 column chromatographyElute from affinity column 13,700 21.9 45.1 chromatography Elute fromhydrophobic 10,300 23.4 33.9 column chromatography Elute from affinitycolumn 5,510 29.6 18.1 chromatography Note: The symbol “*” means theα-isomaltosyl-transferring enzyme of the present invention.

The finally purified α-Isomaltosyl-transferring enzyme specimen wasassayed for purify on gel electrophoresis using a 7.5% (w/v)polyacrylamide gel and detected on the gel as a single protein band,i.e., a high purity specimen.

Experiment 1-3

Purification of α-Isomaltosylglucosaccharide-Forming Enzyme

The crude polypeptide having α-isomaltosylglucosaccharide-forming enzymeactivity, obtained in Experiment 1-1, was dialyzed against 10 mM sodiumphosphate buffer (pH 7.0) with 1 M ammonium sulfate. The dialyzedsolution was centrifuged to remove impurities, and subjected 15 tohydrophobic column chromatography using 350 ml of “BUTYL-TOYOPEARL 650M”gel, a hydrophobic gel commercialized by Tosho Corporation, Tokyo,Japan. The enzyme was adsorbed on “BUTYL-TOYOPEARL 650M” gel and, wheneluted with a linear gradient decreasing from 1 M to 0M of ammoniumsulfate, the enzymatic activity was eluted with a linear gradient ofammonium sulfate at about 0.3 M, and fractions with the enzyme activity5 was collected. The collected solution was dialyzed against 10 mMsodium phosphate buffer (pH 7.0) with 1 M ammonium sulfate, and thedialyzed solution was centrifuged to remove impurities, and purified byaffinity chromatography using “SEPHACRYL HR S-200” gel. The amount ofenzyme activity, specific activity and yield of theα-isomaltosylglucosaccharide-forming enzyme in each purification stepare shown in Table 2.

TABLE 2 Enzyme* Specific activity activity of enzyme* Yield Purificationstep (unit) (unit/mg protein) (%) Culture supernatant 9,180 0.14 100Dialyzed solution after 8,440 0.60 91.9 salting out with ammoniumsulfate Elute from ion-exchange 6,620 1.08 72.1 column chromatographyElute from affinity column 4,130 8.83 45.0 chromatography Elute fromhydrophobic 3,310 11.0 36.1 column chromatography Elute from affinitycolumn 2,000 13.4 21.8 chromatography Note: The symbol “*” means theα-isomaltosylglucosaccharide-forming enzyme.

The finally purified α-isomaltosylglucosaccharide-forming enzymespecimen was assayed for purify on gel electrophoresis using a 7.5%(w/v) polyacrylamide gel and detected on the gel as a single proteinband, i.e., a high purity specimen.

Experiment 2

Physicochemical Properties of Polypeptide having anα-Isomaltosyl-Transferring Enzyme Activity

Experiment 2-1

Action

An aqueous solution containing 10 mM of glucose, 6-O-α-glucosylglucose(isomaltose), 6²-O-α-glucosylmaltose (panose),6³-O-α-glucosylmaltotriose (isomaltosylmaltose),6⁴-O-α-glucosylmaltotetraose, or 6⁵-O-α-glucosylmaltopentaose wasprepared as substrate solution. To each of the above substrate solutionwas added two units/mM-substrate of the purifiedα-isomaltosyl-transferring enzyme specimen obtained in Experiment 1-2and incubated at 30° C. and at pH 6.0 for 12 hours. After deionizing byconventional method, the resulting reaction solutions were measured forsaccharide composition on HPLC using “MCI GEL CK04SS”, a columncommercialized by Mitsubishi Chemical Industries, Ltd., Tokyo, Japan, ata column temperature of 80° C. and a flow rate of 0.4 ml/min of water,and using a detector “RI-8012”, a differential refractometercommercialized by Tosho Corporation, Tokyo, Japan. The results are shownin Table 3.

TABLE 3 Saccharide Content Substrate in the reaction mixture (%) GlucoseGlucose 100 6-O-α-Glucosylglucose 6-O-α-Glucosylglucose 1006²-O-α-Glucosylmaltose Glucose 32.2 Isomaltose 2.16²-O-a-Glucosylmaltose 4.6 Cyclotetrasaccharide 43.5 Isomaltosylpanose4.8 Isomaltosypanoside 1.8 others 11.0 6³-O-α-GlucosylmaltotrioseMaltose 50.6 Isomaltose 2.0 6³-O-α-Glucosylmaltotriose 4.2Cyclotetrasaccharide 30.8 others 12.4 6⁴-O-α- Isomaltose 1.9Glucosylmaltotetraose Maltotriose 60.7 Cyclotetrasaccharide 25.66⁴-O-α-Glucosylmaltotetraose 3.4 others 8.4 6⁵-O-α- Isomaltose 1.6Glucosylmaltopentaose Maltotetraose 66.5 Cyclotetrasaccharide 18.26⁵-O-α-Glucosylmaltopentaose 4.3 others 9.4

In Table 3, isomaltosylpanose means two forms of saccharides having thestructure of structural formula 1 or 2, and isomaltosylpanoside is asaccharide having the structure of Formula 3.α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→4)-α-D-Glcp  Formula1α-D-Glcp-(1→6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→6)-α-D-Glcp-(1→4)-α-D-Glcp  Formula2Formula 3

As evident from the results in Table 3, it was revealed that thepolypeptide having α-isomaltosyl-transferring enzyme activity fromBacillus globisporus C11 acted on saccharides with a glucosepolymerization degree of 3 or higher and having both the α-1,6-glucosyllinkage at their non-reducing end and the α-1,4 glucosidic linkage otherthan the linkage at the non-reducing end such as 6²-O-α-glucosylmaltose,6³-O-α-glucosylmaltotriose, 6⁴-O-a-glucosylmaltotetraose, and6⁵-O-α-glucosylmaltopentaose, and produced mainly cyclotetrasaccharideand maltooligosaccharide which decreased a glucose polymerization degreeof 2 from the substrate. In addition to cyclotetrasaccharide,maltooligosaccharide which decreased a glucose polymerization degree of2 from the substrate, and the remaining substrate, trace isomaltoseconsidered to be a hydrolyzed product and other saccharide which differsfrom cyclotetrasaccharide, and considered to be a glucosyltransferproduct were detected in the reaction mixture. The yield ofcyclotetrasaccharide in dry basis from each substrates, i.e.,6²-O-α-glucosylmaltose, 6³-O-α-glucosylmaltotriose,6⁴-O-a-glucosylmaltotetraose and 6⁵-O-α-glucosylmaltopentaose, were43.5%, 30.8%, 25.6% and 18.2%, respectively. No product was detectedfrom glucose and 6-O-α-glucosylglucose.

Experiment 2-2

N-terminal Amino Acid Sequence

The polypeptide having α-isomaltosyl-transferring enzyme activity had anamino acid sequence of SEQ ID NO:5 at the N-terminal side when the aminoacid sequence was analyzed by “gas-phase protein sequencer model 473A”,an apparatus of Applied Biosystems, 850 Lincoln Centre Drive, FosterCity, U.S.A.

Experiment 2-3

Partial Amino Acid Sequence

A part of a purified specimen of polypeptide havingα-isomaltosyl-transferring enzyme activity, obtained in Experiment 1-2,was dialyzed against 10 mM Tris-HCl buffer (pH 9.0) at 4° C. for 18hours, and the dialyzed solution was diluted with a fresh preparation ofthe same buffer to give a concentration of about one mg/ml. Onemilliliter of the diluted solution as a test sample was admixed with 10μg of “Lysyl Endopeptidase” commercialized by Wako Pure Chemicals, Ltd,Tokyo, Japan, and incubated at 30° C. for 22 hours to form peptides. Theresulting hydrolyzate was subjected to HPLC to separate the peptidesusing “μ-BONDAPAK C18 column”, having a diameter of 2.1 mm and a lengthof 150 mm, a product of Waters Chromatography Div., MILLIPORE Corp.,Milford, USA, pre-equilibrated with 0.1% (v/v) trifluoroacetatecontaining 8% (v/v) acetonitrile, at a flow rate of 0.9 ml/min and atambient temperature, and using a linear gradient of acetonitrileincreasing from 8% (v/v) to 40% (v/v) in 0.1% (v/v) trifluoroacetateover 120 min. Peptide fragments eluted from the column were detected bymonitoring the absorbance at a wavelength of 210 nm. Peptide fractionswith a retention time of about 22 min, about 38 min, about 40 min, about63 min and about 71 min were separately collected and dried in vacuo andthen dissolved in a solution of 0. 1% (v/v) trifluoroacetate and 50%(v/v) acetonitrile. Five peptide fragments were obtained, and eachpeptide fragments had amino acid sequences of SEQ ID NO:6 to 10 whenthese amino acid sequences were analyzed according to the methoddescribed in Experiment 2-2.

Experiment 2-4

Molecular Weight

When a purified specimen of polypeptide havingα-isomaltosyl-transferring enzyme activity, obtained by the method inExperiment 1-2, was subjected to SDS-PAGE according to the methodreported by U. K. Laemmli in Nature, Vol. 227, pp. 680–685 (1970), asingle protein band having the enzymatic activity was observed at theposition corresponding to the molecular weight of about 82,000 to122,000 daltons. Molecular weight markers used in this experiment weremyosin (200,000 daltons), β-galactosidase (116,250 daltons),phosphorylase B (97,400 daltons), serum albumin (66,200 daltons) andovalbumin (45,000 daltons).

Experiment 2-5

Optimum Temperature

As shown in FIG. 1, when a purified specimen of polypeptide havingα-isomaltosyl-transferring enzyme activity, obtained by the method inExperiment 1-2, was acted on the substrate at various temperatures for30 min by conventional method, the polypeptide had an optimumtemperature at about 50° C.

Experiment 2-6

Optimum pH

As shown in FIG. 2, when a purified specimen of polypeptide havingα-isomaltosyl-transferring enzyme activity, obtained by the method inExperiment 1-2, was acted on the substrate in MacIlvaine buffer ofvarious pHs at 35° C. for 30 min by conventional method, the polypeptidehad an optimum pH at about 5.5 to 6.0.

Experiment 2-7

Thermal Stability

As shown in FIG. 3, when a purified specimen of polypeptide havingα-isomaltosyl-transferring enzyme activity, obtained by the method inExperiment 1-2, was incubated in 20 mM acetate buffer (pH 6.0) atvarious temperatures for 60 min by conventional method, the polypeptidehad thermal stability of up to about 40° C.

Experiment 2-8

pH Stability

As shown in FIG. 4, when a purified specimen of polypeptide havingα-isomaltosyl-transferring enzyme activity, obtained by the method inExperiment 1-2, was in MacIlvaine buffer or 50 mM disodiumcarbonate-sodium bicarbonate buffer of various pHs at 4° C. for 24 hoursby conventional method, the polypeptide had pH stability of about 4.5 toabout 9.0.

Experiment 3

Polypeptide from Bacillus globisporus N75

Experiment3-1

Preparation of Crude Polypeptide

A liquid culture medium consisting 4% (w/v) of “PINE-DEX #4”, a partialstarch hydrolyzate, 1.8% (w/v) of “ASAHIMEAST”, a yeast extract, 0.1%(w/v) of dipotassium phosphate, 0.06% (w/v) of sodium phosphatedodeca-hydrate, 0.05% (w/v) of magnesium sulfate hepta-hydrate, andwater was placed in 500-ml Erlenmeyer flasks in a respective amount of100 ml, sterilized by autoclaving at 121° C. for 20 min, cooled andseeded with Bacillus globisporus N75 strain, FERM BP-7591, followed byculturing under rotary-shaking conditions at 27° C. and 230 rpm for 48hours for a seed culture.

About 20 L of a fresh preparation of the same liquid culture medium asused in the above seed culture were placed in a 30 L fermentor,sterilized by heating, and then cooled to 27° C. and inoculated with 1%(v/v) of the seed culture, followed by culturing at 27° C. and pH 6.0 to8.0 for 48 hours under aeration-agitation conditions. The resultantculture, having about 1.1 units/ml of α-isomaltosyl-transferring enzyme,was centrifuged at 10,000 rpm for 30 min to obtain about 18 L ofsupernatant. Measurement of the supernatant revealed that it had about1.1 units/ml of α-isomaltosyl-transferring enzyme activity, i.e., atotal enzyme activity of about 19,800 units; about 0.33 units/ml ofα-isomaltosylglucosaccharide-forming enzyme activity, i.e., a totalenzyme activity of about 5,490 units. It was revealed that both enzymeswere secretion polypeptides detected in the culture supernatant.

About 18 L of the culture supernatant described above was salted outwith 60% saturated ammonium sulfate solution and allowed to stand at 4°C. for 24 hours, and the formed precipitates were collected bycentrifugation at 10,000 rpm for 30 min, dissolved in 10 mM Tris-HClbuffer (pH 8.3), and dialyzed against the same buffer to obtain about450 ml of crude enzyme solution. The crude enzyme solution had about15,700 units of α-isomaltosyl-transferring enzyme activity and 4,710units of α-isomaltosylglucosaccharide-forming enzyme activity. The crudeenzyme solution was subjected to ion-exchange column chromatographyusing “SEPABEADS FP-DA13” gel, disclosed in Experiment 1-1.α-Isomaltosyl-transferring enzyme was eluted as non-adsorbed fractionwithout adsorbing on “SEPABEADS FP-DA13” gel, andα-isomaltosylglucosaccharide-forming enzyme was adsorbed on “SEPABEADSFP-DA13” gel. Subsequently, α-isomaltosylglucosaccharide-forming enzymewas eluted with a linear gradient of increasing from 0 M to 1 M ofsodium chloride, where the enzyme was eluted with the linear gradient ofsodium chloride at a concentration of about 0.25 M. Therefore, fractionswith α-isomaltosyl-transferring enzyme and withα-isomaltosylglucosaccharide-forming enzyme were separately collected ascrude polypeptide having α-isomaltosyl-transferring enzyme activity andthat having α-isomaltosylglucosaccharide-forming enzyme activity,respectively.

Further, the polypeptide having α-isomaltosyl-transferring enzyme andthat having α-isomaltosylglucosaccharide-forming enzyme were separatelypurified and prepared by the methods described in the below.

Experiment 3-2

Purification of a Polypeptide having an α-Isomaltosyl-TransferringEnzyme Acticity

The crude polypeptide having α-isomaltosyl-transferring activity,obtained in Experiment 3-1, was dialyzed against 10 mM sodium phosphatebuffer (pH 7.0) with 1 M ammonium sulfate. The dialyzed solution wascentrifuged to remove impurities, and subjected to affinity columnchromatography using 500 ml of “SEPHACRYL HR S-200” gel, a gelcommercialized by Amersham Corp., Div. Amersham International, Arlingtonheights, Ill., USA. The polypeptide was adsorbed on “SEPHACRYL HR S-200”gel and, when eluted with a linear gradient decreasing from 1 M to 0 Mof ammonium sulfate, the enzymatic activity was eluted with a lineargradient of ammonium sulfate at about 0.3 M, and fractions with theenzyme activity was collected. The collected solution was dialyzedagainst 10 mM sodium phosphate buffer (pH 7.0) with 1 M ammoniumsulfate, and the dialyzed solution was centrifuged to remove impurities,and purified by hydrophobic column chromatography using “BUTYL-TOYOPEARL650M” gel, a hydrophobic gel commercialized by Tosho Corporation, Tokyo,Japan. The polypeptide was adsorbed on “BUTYL-TOYOPEARL 650M” gel and,when eluted with a linear gradient decreasing from 1 M to 0M of ammoniumsulfate, the enzymatic activity was eluted with a linear gradient ofammonium sulfate at about 0.3 M, and fractions with the enzyme activitywas collected. The collected solution was dialyzed against 10 mMTris-HCl buffer (pH 8.0), and the dialyzed solution was centrifuged toremove impurities, and purified by ion-exchange column chromatographyusing 380 ml of “SuperQ-TOYOPEARL 650C” gel, a ion-exchange gelcommercialized by Tosho Corporation, Tokyo, Japan. The polypeptide waseluted as non-adsorbed fraction without adsorbing on “SuperQ-TOYOPEARL650C” gel. The purified polypeptide specimen havingα-isomaltosyl-transferring enzyme activity was obtained by collectingthe fractions. The amount of enzyme activity, specific activity andyield of the α-isomaltosyl-transferring enzyme in each purification stepare shown in Table 4.

TABLE 4 Enzyme* Specific activity activity of enzyme* Yield Purificationstep (unit) (unit/mg protein) (%) Culture supernatant 19,000 0.33 100Dialyzed solution after 15,700 0.64 82.6 salting out with ammoniumsulfate Elute from ion-exchange 12,400 3.56 65.3 column chromatographyElute from affinity column 8,320 11.7 43.8 chromatography Elute fromhydrophobic 4,830 15.2 25.4 column chromatography Elute fromion-exchange 3,850 22.6 20.3 column chromatography Note: The symbol “*”means the α-isomaltosyl-transferring enzyme of the present invention.

The finally purified α-isomaltosyl-transferring enzyme specimen wasassayed for purity on gel electrophoresis using a 7.5% (w/v)polyacrylamide gel and detected on the gel as a single protein band,i.e., a high purity specimen.

Experiment 3-3

Purification of α-Isomaltosylglucosaccharide-Forming Enzyme

The crude polypeptide having α-isomaltosylglucosaccharide-forming enzymeactivity, obtained in Experiment 3-1, was dialyzed against 10 mM sodiumphosphate buffer (pH 7.0) with 1 M ammonium sulfate. The dialyzedsolution was centrifuged to remove impurities, and subjected to affinitycolumn chromatography using 500 ml of “SEPHACRYL HR S-200” gel, a gelcommercialized by Amersham Corp., Div. Amersham International, Arlingtonheights, Ill., USA. The enzyme was adsorbed on “SEPHACRYL HR S-200” geland, when sequentially eluted with a linear gradient decreasing from 1 Mto 0 M of ammonium sulfate and a linear gradient increasing from 0 mM to100 mM of maltotetraose, the enzymatic activity was eluted with a lineargradient of maltotetraose at about 30 mM, and fractions with the enzymeactivity was collected. The collected solution was dialyzed against 10mM sodium phosphate buffer (pH 7.0) with 1 M ammonium sulfate, and thedialyzed solution was centrifuged to remove impurities, and purified byhydrophobic column chromatography using 350 ml of “BUTYL-TOYOPEARL 650M”gel, a hydrophobic gel commercialized by Tosho Corporation, Tokyo,Japan. The enzyme was adsorbed on “BUTYL-TOYOPEARL 650M” gel and, wheneluted with a linear gradient decreasing from 1 M to 0 M of ammoniumsulfate, the enzymatic activity was eluted with a linear gradient ofammonium sulfate at about 0.3 M, and fractions with the enzyme activitywas collected. The collected solution was dialyzed against 10 mM sodiumphosphate buffer (pH 7.0) with 1 M ammonium sulfate, and the dialyzedsolution was centrifuged to remove impurities, and purified by affinitychromatography using “SEPHACRLY HR S-200” gel. The amount of enzymeactivity, specific activity and yield of theα-isomaltosylglucosaccharide-forming enzyme in each purification stepare shown in Table 5.

TABLE 5 Enzyme* Specific activity activity of enzyme* Yield Purificationstep (unit) (unit/mg protein) (%) Culture supernatant 5,940 0.10 100Dialyzed solution after 4,710 0.19 79.3 salting out with ammoniumsulfate Elute from ion-exchange 3,200 2.12 53.9 column chromatographyElute from affinity column 2,210 7.55 37.2 chromatography Elute fromhydrophobic 1,720 10.1 29.0 column chromatography Elute from affinitycolumn 1,320 12.5 22.2 chromatography Note: The symbol “*” means theα-isomaltosylglucosaccharide-forming enzyme.

The finally purified α-isomaltosylglucosaccharide-forming enzymespecimen was assayed for purity on gel electrophoresis using a 7.5%(w/v) polyacrylamide gel and detected on the gel as a single proteinband, i.e., a high purity specimen.

Experiment 4

Physicochemical Properties of Polypeptide having anα-Isomaltosyl-Transferring Enzyme Activity

Experiment 2-1

Action

An aqueous solution containing 10 mM of glucose, 6-O-α-glucosylglucose(isomaltose), 6²-O-α-glucosylmaltose (panose),6³-O-α-glucosylmaltotriose (isomaltosylmaltose),6⁴-O-αglucosylmaltotetraose, or 6⁵-O-α-glucosylmaltopentaose wasprepared as substrate solution. To each of the above substrate solutionswas added two units/mM-substrate of the purifiedα-isomaltosyl-transferring enzyme specimen obtained in Experiment 3-2and incubated at 30° C. and at pH 6.0 for 12 hours. After deionizing byconventional method, the resulting reaction solutions were measured forsaccharide composition on HPLC, disclosed in Experiment 2-1. The resultsare shown in Table 6.

TABLE 6 Saccharide Content Substrate in the reaction mixture (%) GlucoseGlucose 100 6-O-α-Glucosylglucose 6-O-α-Glucosylglucose 1006²-O-α-Glucosylglucose Glucose 31.8 Isomaltose 2.06²-O-a-Glucosylglucose 4.4 Cyclotetrasaccharide 43.2 Isomaltosylpanose6.5 Isomaltosypanoside 2.4 others 9.7 6³-O-α-Glucosylglucose Maltose50.3 Isomaltose 1.9 6³-O-α-Glucosylglucose 4.5 Cyclotetrasaccharide 30.9others 12.4 6⁴-O-α-Glucosylglucose Isomaltose 1.5 Maltotriose 60.9Cyclotetrasaccharide 25.8 6⁴-O-α-Glucosylglucose 3.2 others 8.66⁵-O-α-Glucosylglucose Isomaltose 1.4 Maltotetraose 66.6Cyclotetrasaccharide 18.7 6⁵-O-α-Glucosylglucose 4.2 others 9.1

As evident from the results in Table 6, it was revealed that thepolypeptide having α-isomaltosyl-transferring activity from Bacillusglobisporus N75 acted on saccharides with a glucose polymerizationdegree of 3 or higher and having both the α-1,6-glucosidic linkage as alinkage at the non-reducing end and the α-1,4 glucosidic linkage otherthan the linkage at the non-reducing ends such as6²-O-α-glucosylmaltose, 6³-O-α-glucosylmaltotriose,6⁴-O-α-glucosylmaltotetraose, and 6⁵-O-α-glucosylmaltopentaose, andproduced mainly cyclotetrasaccharide and maltooligosaccharides whichdecreased a glucose polymrization degree of 2 from the substrate. Inaddition to cyclotetrasaccharide, maltooligosaccharide which decreased aglucose polymerization degree of 2 from the substrate, and the remainingsubstrate, trace isomaltose considered to be a hydrolyzed product andother saccharide which differs from cyclotetrasaccharide, and consideredto be a glucosyltransfer product were detected in the reaction mixture.The yield of cyclotetrasaccharide in dry basis from 6²-O-α-glucosylmaltose, 6³-O-α-glucosylmaltotriose,6⁴-O-α-glucosylmaltotetraose and 6⁵-O-α-glucosylmaltopentaose were43.2%, 30.9%, 25.8% and 18.7%, respectively. No product was detectedfrom 6-O-α-glucosylglucose.

Experiment 4-2

N-Terminal Amino Acid Sequence

The polypeptide having α-isomaltosyl-transferring enzyme activity,prepared in Experiment 3-2, had an amino acid sequence of SEQ ID NO:5 atN-terminal side when the amino acid sequence was analyzed by “proteinsequencer model 473A”, an apparatus of Applied Biosystems, 850 LincolnCentre Drive, Foster City, U.S.A.

Experiment 4-3

Partial Amino Acid Sequence

A part of a purified specimen of polypeptide havingα-isomaltosyl-transferring activity, obtained in Experiment 3-2, wasdialyzed against 10 mM Tris-HCl buffer (pH 9.0) at 4° C., and thedialyzed solution was diluted with a fresh preparation of the samebuffer to give a concentration of about one mg/ml. One milliliter of thediluted solution as a test sample was admixed with 10 μg of “LysylEndopeptidase” commercialized by Wako Pure Chemicals, Ltd, Tokyo, Japan,and incubated at 30° C. for 22 hours to form peptides. The resultingpartial hydrolyzate was subjected to HPLC to separate the peptides using“μ-BONDASPHERE C18 column”, having a diameter of 3.9 mm and a length of150 mm, a product of Waters Chromatography Div., MILLIPORE Corp.,Milford, USA, pre-equilibrated with 0.1% (v/v) trifluoroacetatecontaining 4% (v/v) acetonitrile, at a flow rate of 0.9 ml/min and atambient temperature, and using a linear gradient of acetonitrileincreasing from 8% (v/v) to 42.4% (v/v) in 0.1% (v/v) trifluoroacetateover 90 min. Peptide fragments eluted from the column were detected bymonitoring the absorbance at a wavelength of 210 nm. Peptide fractionswith a retention time of about 21 min, about 38 min, about 56 min, andabout 69 min were separately collected and dried in vacuo and thendissolved in a solution of 0.1% (v/v) trifluoroacetate and 50% (v/v)acetonitrile. Five peptide fragments were obtained, and each peptidefragment had amino acid sequences of SEQ ID NO:8 and 11 to 14 when theseamino acid sequences were analyzed according to the method described inExperiment 2-2.

Experiment 4-4

Molecular Weight

When a purified specimen of polypeptide havingα-isomaltosyl-transferring activity, obtained by the method inExperiment 3-2, was subjected to SDS-PAGE according to the methoddisclosed in Experiment 2-4, a single protein band having the enzymaticactivity was observed at the position corresponding to the molecularweight of about 92,000 to 132,000 daltons in comparison with molecularmarkers, commercialized by Bio-Rad Laboratories, Hercules, Calif. 94547,U.S.A., and subjected to SDS-PAGE at the same time.

Experiment 4-5

Octimum Temperature

A purified specimen of polypeptide having α-isomaltosyl-transferringactivity, obtained by the method in Experiment 3-2, was acted on thesubstrate in 20 mM acetate buffer (pH6.0) at various temperatures for 30min, according to the assay method of α-isomaltosyl-transferring enzymedisclosed in Experiment 1-1. As shown in FIG. 5, the polypeptide had anoptium temperature at about 50 ° C.

Experiment 4-6

Octimum pH

A purified specimen of polypeptide having α-isomaltosyl-transferringactivity, obtained by the method in Experiment 3-2, was acted on thesubstrate in MacIlvaine buffer of various pHs at 35° C. for 30 minaccording to the assay method of α-isomaltosyl-transferring enzymedisclosed in Experiment 1-1. As shown inm FIG. 6, the polypeptide had anoctimum pH at about 6.0

Experiment 4-7

Thermal Stability

A purified specimen of polypeptide having α-isomaltosyl-transferringactivity, obtained by the method in Experiment 3-2, was incubated in 20mM acetate buffer (pH6.0) at various temperatures for 60 min accordingto the assay method of α-isomaltosyl-transferring enzyme disclosed inExperiment 1-1. As shown in FIG. 7, the polypeptide had thermalstability of up to about 45° C.

Experiment 4-8

pH Stability

A purified specimen of polypeptide having α-isomaltosyl-transferringactivity, obtained by the method in Experiment 3-2, was incubated inMacIlvaine buffer or 50 mM disodium carbonate-sodium bicarbonate bufferof various pHs at 4° C. for 24 hours according to the assay method ofα-isomaltosyl-transferring enzyme disclosed in Experiment 1-1. As shownin FIG. 8, the polypeptide had pH stability of about 4.5 to about 10.

Experiment 5

Recombinant DNA Containing a DNA Encoding a Polypeptide from Bacillusglobisporus C11 and Transformant

Experiment 5-1

Preparation of Chromosonal DNA from Bacillus globisporus C11

A liquid culture medium consisting 2% (w/v) of “PINE-DEX #4”, a partialstarch hydrolyzate, 1.0% (w/v) of “ASAHIMEAST”, a yeast extract, 0.1%(w/v) of dipotassium phosphate, 0.06% (w/v) of sodium phosphatedodeca-hydrate, 0.05% (w/v) of magnesium sulfate hepta-hydrate, andwater was placed in 500-ml Erlenmyer flasks in a respective amount of100 ml, sterilized by autoclaving at 121° C. for 20 min, cooled andinoculated with Bacillus globisporus C11, FERM BP-7144, followed byculturing under rotary-shaking conditions at 27° C. and 230 rpm for 24hours.The cells collected from the culture by centrifugation weresuspended in TES buffer (pH 8.0), the suspended solution was admixedwith lysozyme to give a concentration of 0.05% (w/v), and incubated at37° C. for 30 min. After freezing the lysate at −80° C. for one hour,the lysate was added with TES buffer (pH 9.0)and heated to 60° C. Thesolution was added with a mixture of TES buffer and phenol, and wasvigorously shaken for five minute in an ice bath, and the supernatantwas collected by centrifugation. The supernatant was added to twice thevolume of cold ethanol, and the resulting crude precipitate wascollected as a crude chromosomal DNA. The crude chromosomal DNA wasdissolved in SSC buffer (pH 7.1), and admixed with 7.5 μg ofribonuclease and 125 μg of proteinase, and incubated 37° C. for onehour. The chromosomal DNA was extracted from the reactant by addingchloroform/isoamylalcohol mixture, then added cold ethanol, and theresulting precipitate containing chromosomal DNA was collected. Thepurified chromosomal DNA, obtained according to the method describedabove, was dissolved in SSC buffer (pH 7.1) to give a concentration ofabout one mg/ml and frozen at −80° C.

Experiment 5-2

Preparation of a Recombinant DNA, pBGC1 and a Transformant, BGC1

One milliliter of purified chromosomal DNA solution, prepared by themethod in Experiment 5-1, was admixed with about 35 units of arestriction enzyme, Sau 3AI, and incubated at 37□ for 20 min for partialdigestion of the chromosomal DNA. The resulting DNA fragmentscorresponding to about 2,000 to 6,000 base pairs were collected bysucrose density-gradient centrifugation. A plasmid vector, Bluescript IISK(+), commercialized by Stratagene Cloning System, was completelydigested with a restriction enzyme, Bam HI by conventional method. Arecombinant DNA was obtained by ligating 0.5 μg of the digested plasmidvector with about 5 μg of the DNA fragments prepared before by using a“DNA ligation kit”, commercialized by Takara Shuzo Co., Ltd., accordingto the method described in a document attached with the kit. Then, agene library was prepared by transforming 100 μl portion of thecompetent cell, “Epicurian Coli XL2-Blue”, commercialized by StratageneCloning System, with the recombinant DNA by conventional competent cellmethod. The transformants thus obtained as gene library were inoculatedinto a fresh agar plate medium (pH 7.0) containing 10 g/L of tryptone, 5g/L of yeast extract, 5 g/L of sodium chloride, 100 mg/L of ampicillinsodium salt, and 50 mg/L of 5-bromo-4-chloro-3-indolyl-β-galactoside,and incubated at 37□ for 24 hours. About five thousand white coloniesgrown on the plate were transferred to and fixed on a nylon membrane,“Hybond-N+”, commercialized by Amasham Bioscience K.K. Anoligonucleotide having a nucleotide sequence of“5′-AAYTGGTGGATGWSNAA-3′” (SEQ ID NO:17) was chemically synthesized onthe bases of an amino acid sequence of first to sixth of SEQ ID NO:8,which disclosed by the method in Experiment 2-3. A synthetic DNA(probe 1) was obtained by labeling the oligonucleotide with radioisotopeusing [γ-³²P]ATP and T4 polynucleotide kinase according to theconventional method. Subsequently, four types of transformants showingremarkable hybridization with probe 1 were selected from the coloniesfixed on the nylon membrane obtained before, using conventional colonyhybridization. The recombinant DNAs were collected from these four typesof transformants by conventional method. On the other hand, probe 2having the nucleotide sequence of “5′-GTNTTYAAYCARTAYAA-3′” (SEQ IDNO:18) was chemically synthesized based on a amino acid sequence ofninth to fourteenth of SEQ ID NO:7 and labeled with radioisotope in thesame manner. The recombinant DNAs obtained and probe 2 were used forconventional southern-hybridization, and a recombinant DNA showing aremarkable hybridization with probe 2 was selected. A transformant thusselected was named “BGC1”. According to the conventional method, thetransformant, BGC1 was inoculated into L-broth medium (pH 7.0)containing 100 μg/ml of ampicillin sodium salt, and cultured underrotary-shaking conditions at 37□ for 24 hours. After the completion ofthe culture, cells were collected by centrifugation, and the recombinantDNA was extracted from the cells by conventional alkaline-SDS method.When the nucleotide sequence of the recombinant DNA was analyzed byconventional dideoxy method, it was revealed that the recombinant DNAcontained a DNA having the nucleotide sequence of SEQ ID NO:15, 3,869base pairs, which originated from Bacillus globisporus C11 (FERMBP-7144). In the recombinant DNA, a DNA having the nucleotide sequenceof SEQ ID NO:15 was shown in FIG. 9 with the part of black-bold line,and was ligated at downstream of recognition site of a restrictionenzyme, Xba I.

The amino acid sequence deduced from the nucleotide sequence is as shownin parallel in SEQ ID NO:15. The amino acid sequence was compared withamino acid sequences of polypeptide having α-isomaltosyl-transferringenzyme activity, i.e., the N-terminal amino acid sequence of SEQ ID NO:5disclosed by the method in Experiment 2-2 and the internal partial aminoacid sequences of SEQ ID NO:6 to 10 disclose by the method in Experiment2-3. An amino acid sequence of SEQ ID NO:5 was completely identical withthat of 30th to 48th of the amino acid sequence shown in parallel in SEQID NO:15. Amino acid sequences of SEQ ID NO:6, 7, 8, 9, and 10 werecompletely identical with those of 584th to 597th, 292nd to 305th, 545thto 550th, 66th to 77th, and 390th to 400th of the amino acid sequenceshown in parallel in SEQ ID NO:15, respectively. These results indicatethat the polypeptide having α-isomaltosyl-transferring enzyme activitycontains the amino acid sequence of SEQ ID NO:1, and that thepolypeptide is encoded by the DNA having the nucleotide sequence of SEQID NO:3 in the case of Bacillus globisporus C11 (FERM BP-7144). An aminoacid sequence of the first to 29th of that showing in parallel in SEQ IDNO:15 was presumed to be a secretion signal sequence of the polypeptide.According to the results described above, it was revealed that theprecursor peptide of the polypeptide before secretion had the amino acidsequence shown in parallel in SEQ ID NO:15, and the amino acid sequencewas encoded by the nucleotide sequence of SEQ ID NO:15. The recombinantDNA prepared and confirmed the nucleotide sequence as described abovewas named “pBGC1”.

Experiment 6

Preparation of a Recombinant DNA Containing a DNA Encoding Polypeptidefrom Bacillus globisporus N75 and a Transformant

Experiment 6-1

Preparation of Chromosomal DNA from Bacillus globisporus N75

A liquid culture medium consisting 2% (w/v) of “PINE-DEX #4”, a partialstarch hydrolyzate, 1.0% (w/v) of “ASAHIMEAST”, a yeast extract, 0.1%(w/v) of dipotassium phosphate, 0.06% (w/v) of sodium phosphatedodeca-hydrate, 0.05% (w/v) of magnesium sulfate hepta-hydrate, andwater was placed in 500-ml Erlenmeyer flasks in a respective amount of100 ml, sterilized by autoclaving at 121° C. for 20 min, cooled andinoculated with Bacillus globisporus N75, FERM BP-7591, followed byculturing under rotary-shaking conditions at 27° C. and 230 rpm for 24hours. The cells collected from the culture by centrifugation weresuspended in TES buffer (pH 8.0), the suspended solution was admixedwith lysozyme to give a concentration of 0.05% (w/v), and incubated at37° C. for 30 min. After freezing the lysate at −80° C. for one hour,the lysate was added with TES buffer (pH 9.0)and heated to 60° C. Thesolution was added with a mixture of TES buffer and phenol, and wasvigorously shook for five minute in an ice bath, and the supernatant wascollected by centrifugation. The supernatant was added twice volume ofcold ethanol, and resulting Crude precipitate was collected as crudechromosomal DNA. The crude chromosomal DNA was dissolved in SSC buffer(pH 7.1), and admixed with 7.5 μg of ribonuclease and 125 μg ofproteinase, and incubated 37° C. for one hour. The chromosomal DNA wasextracted from the reactant by adding chloroform/isoamylalcohol mixture,then added cold ethanol, and the resulting precipitate containingchromosomal DNA was collected. The purified chromosomal DNA, obtainedaccording to the method described above, was dissolved in SSC buffer (pH7.1) to give a concentration of about one mg/ml and frozen at −80° C.

Experiment 6-2

Preparation of a Recombinant DNA, pBGN1 and a Transformant, BGN1

One hundred μl (0.1 ml) of purified chromosomal DNA solution, preparedby the method in Experiment 6-1, was admixed with about 100 units of arestriction enzyme, Sac I, and incubated at 37□ for 6 hours to digestthe chromosomal DNA. The resulting DNA fragments were separated byagarose gel electrophoresis, and DNA fragments corresponding to about3,000 to 7,000 base pairs were collected using a DNA purification kit,“GENECLEAN II KIT”, commercialized by Quantum Biotechnologies, Carlsbad,Calif. 92008, U.S.A., according to the method described in a documentattached with the kit. A plasmid vector, Bluescript II SK(+),commercialized by Stratagene Cloning System, was completely digestedwith a restriction enzyme, Sac I. A recombinant DNA was obtained byligating 0.5 μg of the digested plasmid vector with about 5 μg of theDNA fragments prepared before by using a “DNA ligation kit”,commercialized by Takara Shuzo Co., Ltd., according to the methoddescribed in a document attached with the kit. Then, a gene library wasprepared by transforming 100 μl portion of the competent cell,“Epicurian Coli XL2-Blue”, commercialized by Stratagene Cloning System,with the recombinant DNA by conventional competent cell method. Thetransformants thus obtained as gene library were inoculated into a freshagar plate medium (pH 7.0) containing 10 g/L of tryptone, 5 g/L of yeastextract, 5 g/L of sodium chloride, 100 mg/L of ampicillin sodium salt,and 50 mg/L of 5-bromo-4-chloro-3-indolyl-β-galactoside, and incubatedat 37° C. for 24 hours. About four thousand white colonies grown on theplate were transferred to and fixed on a nylon membrane, “Hybond-N+”,commercialized by Amasham Bioscience K.K. An oligonucleotide having anucleotide sequence of “5′-AAYTGGTGGATGWSNAA-3′” (SEQ ID NO:17) waschemically synthesized on the bases of an amino acid sequence of firstto sixth of SEQ ID NO:8, which disclosed by the method in Experiment2-3. A synthetic DNA (probe 1) was obtained by labeling theoligonucleotide with radioisotope using [γ-³²P]ATP and T4 polynucleotidekinase according to the conventional method. Subsequently, two types oftransformant showing remarkable hybridization with probe 1 were selectedfrom the colonies fixed on the nylon membrane obtained before, usingconventional colony hybridization. The recombinant DNAs were collectedfrom these two types of transformant by conventional method. On theother hand, probe 2 having the nucleotide sequence of“5′-GAYTGGATHGAYTTYTGGTTYGG-3′” (SEQ ID NO:19) was chemicallysynthesized based on a amino acid sequence of eighth to fifteenth of SEQID NO:14 and labeled with radioisotope in the same manner. Therecombinant DNAs obtained and probe 2 were used for conventionalsouthern-hybridization, and a recombinant DNA showing a remarkablehybridization with probe 2 was selected. A transformant thus selectedwas named “BGN1”. According to the conventional method, thetransformant, BGN1 was inoculated into L-broth medium (pH 7.0)containing 100 μg/ml of ampicillin sodium salt, and cultured underrotary-shaking conditions at 37° C. for 24 hours. After the completionof the culture, cells were collected by centrifugation, and therecombinant DNA was extracted from the cells by conventionalalkaline-SDS method. When the nucleotide sequence of the recombinant DNAwas analyzed by conventional dideoxy method, it was revealed that therecombinant DNA contained a DNA having the nucleotide sequence of SEQ IDNO:16, 4,986 base pairs, which originated from Bacillus globisporus N75(FERM BP-591). In the recombinant DNA, a DNA having the nucleotidesequence of SEQ ID NO:16 was shown in FIG. 10 with the part ofblack-bold line, and was ligated at downstream of recognition site of arestriction enzyme, Sac I.

The amino acid sequence deduced from the nucleotide sequence is as shownin parallel in SEQ ID NO:16. The amino acid sequence was compared withamino acid sequences of polypeptide having α-isomaltosyl-transferringenzyme activity, i.e., the N-terminal amino acid sequence of SEQ ID NO:5disclosed by the method in Experiment 4-2 and the internal partial aminoacid sequences of SEQ ID NO:8 and 11 to 14 disclose by the method inExperiment 4-3. An amino acid sequence of SEQ ID NO:5 was completelyidentical with that of 30th to 48th of the amino acid sequence shown inparallel in SEQ ID NO:16. Amino acid sequences of SEQ ID NO:8, 11, 12,13, and 14 were completely identical with those of 545th to 550th, 565thto 582nd, 66th to 83rd, 390th to 406th, and 790th to 809th of the aminoacid sequence shown in parallel in SEQ ID NO:16, respectively. Theseresults indicate that the polypeptide having α-isomaltosyl-transferringenzyme activity contains the amino acid sequence of SEQ ID NO:2, andthat the polypeptide is encoded by the DNA having the nucleotidesequence of SEQ ID NO:4 in the case of Bacillus globisporus N75 (FERMBP-7591). An amino acid sequence of the first to 29th of that showing inparallel in SEQ ID NO:16 was presumed to be a secretion signal sequenceof the polypeptide. According to the results described above, it wasrevealed that the precursor peptide of the polypeptide before secretionhad the amino acid sequence shown in parallel in SEQ ID NO:16, and theamino acid sequence was encoded by the nucleotide sequence of SEQ IDNO:16. The recombinant DNA prepared and confirmed the nucleotidesequence as described above was named “pBGN1”.

Experiment 7

Production of Polypeptides having α-Isomaltosyl-Transferring EnzymeActivity by Transformants

Experiment 7-1

A Transformant, BGC1

A liquid culture medium consisting 5 g/L of “PINE-DEX #4”, a partialstarch hydrolyzate, 20 g/L of polypeptone, 20 g/L of yeast extract, 1g/L of sodium phosphate dodeca-hydrate, and water was placed in a 500-mlErlenmeyer flask in a amount of 100 ml, sterilized by autoclaving at121° C. for 15 min, and cooled. Then, the liquid medium was sterilelyset to pH 7.0, and sterilely admixed with 10 mg of ampicillin sodiumsalt. A transformant, BGC1, obtained by the method in Experiment 5-2,was inoculated into the above liquid medium, and cultured at 27° C. andfor 48 hours under aeration-agitation conditions. To investigate thelocation of the polypeptide in the culture, cells and supernatant wereseparately collected by conventional centrifugation. In the case of thecells, whole-cell extract, obtained by ultrasonic disruption, andperiplasmic extract, obtained by osmotic shock procedure were preparedseparately. In the case of ultrasonic disruption, cells were suspendedin 10 mM sodium phosphate buffer (pH 7.0), and then disrupted in an icebath using a ultrasonic homogenizer, “model UH-600”, commercialized byMST Corporation, Aichi, japan. In the case of osmotic shock procedure,cells were washed with 10 mM Tris-HCl buffer (pH 7.3) containing 30 mMsodium chloride, and the washed cells were suspended in 33 mM Tris-HClbuffer (pH 7.3) containing 200 g/L of sucrose and 1 mM EDTA, shook at27° C. for 20 min, and then centrifuged to collect the cells.Subsequently, the cells were suspended in 0.5 mM magnesium chloridesolution pre-cooled to about 4° C., and shaken in ice bath for 20 min toextract periplasmic fraction. α-Isomaltosyl-transferring enzymeactivities of culture supernatant, whole-cell extract and periplasmicextract, prepared as described above, were assayed, and those valueswere expressed in terms of the activities/ml-culture, respectively. Theresults are shown in Table 7.

TABLE 7 α-isomaltosyl-transferring enzyme activity Sample(units/ml-culture) Culture supernatant 0.0 Whole-cell extract 3.4Periplasmic extract 3.0

As evident from the results in Table 7, it was revealed that thetransformant, E. coli BGC1 produced the polypeptide havingα-isomaltosyl-transferring enzyme activity of the present inventionintracellularly, and secreted most of it in periplasmic fraction.

As the first control experiment, E. coli XL2-Blue was cultured with thesame conditions in the case of the transformant described above exceptfor the addition of ampicillin, and a supernatant and a cell-extractwere prepared from the culture. As the second control experiment,Bacillus globisporus C11, FERM BP-7144, was cultured with the sameconditions in the case of the transformant described above except forthe addition of ampicillin, and a supernatant and a cell-extract wereprepared from the culture. In the first control experiment, the enzymeactivity was not detected from either of the culture supernatant and thecell-extract. In the second control experiment, the enzyme activity ofthe culture supernatant and the cell-extract were about 1.2 units andabout 0.1 units, respectively, and the total enzyme activity per onemilliliter-culture was about 1.3 units. Compared with the total enzymeactivity, 3.4 units/ml-culture, of the transformant BGC1, the enzymeactivity was evidently low-level values.

The periplasmic fraction was further purified by salting out, dialysisand successive column chromatographies on “SEPABEADS FP-DA13” gel,“SEPHACRYL HR S-200” gel, and “BUTYL-TOYOPEARL 650M” gel according tothe methods described in Experiment 1, and the purified polypeptide wasanalyzed according to the methods described in Experiment 2. As theresults, the molecular weight was about 82,000 to 122,000 daltons bySDS-polyacrylamide gel electrophoresis, the isoelectric point was about5.1 to 6.1 by polyacrylamide gel isoelectrophoresis, the optimumtemperature of α-isomaltosyl-transferring enzyme activity was about 50°C., the optimum pH of the enzyme was about 5.5 to 6.0, the thermalstability was up to about 40° C., and the pH stability was in the rangeof about pH 4.5 to about 9. These physicochemical properties werepractically identical to those of the polypeptide havingα-isomaltosyl-transferring enzyme activity prepared in Experiment 1. Theresults described above indicate that recombinant DNA techniques enableto produce polypeptide having the α-isomaltosyl-transferring enzymeactivity of the present invention stably and in large scale and at arelatively low cost.

Experiment 7-2

A Transformant, BGN1

A liquid culture medium consisting 5 g/L of “PINE-DEX #4”, a partialstarch hydrolyzate, 20 g/L of polypeptone, 20 g/L of yeast extract, 1g/L of sodium phosphate dodec a-hydrate, and water was placed in a500-ml Erlenmeyer flask in a amount of 100 ml, sterilized by autoclavingat 121° C. for 15 min, and cooled. Then, the liquid medium was sterilelyset to pH 7.0, and sterilely admixed with 10 mg of ampicillin sodiumsalt. A transformant, BGN1, obtained by the method in Experiment 6-2,was inoculated into the above liquid medium, and cultured at 27° C. andfor 48 hours under aeration-agitation conditions. To investigate thelocation of the polypeptide in the culture, cells and supernatant wereseparately collected by conventional centrifugation. As described inExperiment 7-1, whole-cell extract, obtained by ultrasonic disruption,and periplasmic extract, obtained by osmotic shock procedure wereprepared separately. α-Isomaltosyl-transferring enzyme activities ofculture supernatant, whole-cell extract and periplasmic extract wereassayed, and those values were expressed in terms of theactivities/ml-culture, respectively. The results are shown in Table 8.

TABLE 8 α-isomaltosyl-transferring enzyme activity Sample(units/ml-culture) Culture supernatant 0.2 Whole-cell extract 3.1Periplasmic extract 2.9

As evident from the results in Table 8, it was revealed that thetransformant, E. coli BGN1 produced the polypeptide havingα-isomaltosyl-transferring enzyme activity of the present inventionintracellularly, and secreted most of it in periplasmic fraction. Theenzyme activity was also detected in culture supernatant.

As the first control experiment, E. coli XL2-Blue was cultured with thesame conditions in the case of the transformant described above exceptfor the addition of ampicillin, and a supernatant and a cell-extractwere prepared from the culture. As the second control experiment,Bacillus globisporus N75, FERM BP-7591, was cultured with the sameconditions in the case of the transformant described above except forthe addition of ampicillin, and a supernatant and a cell-extract wereprepared from the culture. In the first control experiment, the enzymeactivity was not detected from either of the culture supernatant and thecell-extract. In the second control experiment, the enzyme activity ofthe culture supernatant and the cell-extract were about 0.7 units andabout 0.1 units, respectively, and the total enzyme activity per onemilliliter-culture was about 0.8 units. Compared with the total enzymeactivity, 3.3 units/ml-culture, of the transformant BGN1, the enzymeactivity was evidently low-level values.

The periplasmic fraction was further purified by salting out, dialysisand successive column chromatographies on “SEPABEADS FP-DA13” gel,“SEPHACRYL HR S-200” gel, and “BUTYL-TOYOPEARL 650M” gel according tothe methods described in Experiment 3, and the purified polypeptide wasanalyzed according to the methods described in Experiment 4. As theresults, the molecular weight was about 92,000 to 132,000 daltons bySDS-polyacrylamide gel electrophoresis, the isoelectric point was about7.3 to 8.3 by polyacrylamide gel isoelectrophoresis, the optimumtemperature of α-isomaltosyl-transferring enzyme activity was about 50°C., the optimum pH of the enzyme was about 6.0, the thermal stabilitywas up to about 45° C., and the pH stability was in the range of aboutpH 4.5 to about 10. These physicochemical properties were practicallyidentical to those of the polypeptide having α-isomaltosyl-transferringenzyme activity prepared in Experiment 3. The results described aboveindicate that recombinant DNA techniques enable to produce polypeptidehaving the α-isomaltosyl-transferring enzyme activity of the presentinvention stably and in large scale and at a relatively low cost.

As described above, a polypeptide having an activity to form acyclotetrasaccharide from a saccharide with a glucose polymerizationdegree of 3 or higher and bearing both the α-1,6 glucosidic linkage as alinkage at the non-reducing end and the α-1,4 glucosidic linkage otherthan the linkage at the non-reducing end, comprising amino acidsequences of either SEQ ID NO:1 or SEQ ID NO:2, or the amino acidsequences having deletion, replacement or insertion of one or more aminoacids of SEQ ID NO:1 or SEQ ID NO:2, is found as one of the product of along studies by the present inventors, and has unique physicochemicalproperties in comparison with the enzymes ever known. The presentinvention intends to create the polypeptide by applying recombinant DNAtechniques. The following explain the polypeptide of the presentinvention, its production processes and its uses in detail with thereferences of examples.

The polypeptide as referred to in the present invention means the wholepolypeptides which have an activity to form a cyclotetrasaccharide froma saccharide with a glucose polymerization degree of 3 or higher andbearing both the α-1,6 glucosidic linkage as a linkage at thenon-reducing end and the α-1,4 glucosidic linkage other than the linkageat the non-reducing end, and comprises amino acid sequences of eitherSEQ ID NO:1 or SEQ ID NO:2, or the amino acid sequences having deletion,replacement or addition of one or more amino acids of SEQ ID NO:1 or SEQID NO:2. The polypeptide of the present invention usually comprises asolved amino acid sequence, for example, amino acid sequence of SEQ IDNO:1, SEQ ID NO:2 or homologous amino acid sequences of those. Mutantshaving the homologous amino acid sequences with SEQ ID NO:1 or SEQ IDNO:2 can be obtained by deleting, replacing or adding one or more, i.e.,at least one or two, according to the situation, 1–50, 1–30, or 1–10amino acids of SEQ ID NO:1 or SEQ ID NO:2 without altering the inherentphysicochemical properties of the enzyme practically. Even using thesame DNA, the post-translational modification of the polypeptide byextra-/intra-cellular enzymes of host is affected by various conditionssuch as kinds of host, nutrients or composition of culture media,temperatures or pHs for the cultivation of a transformant having theDNA. In such conditions, it is possible to arise some mutants havingdeletion or replacement of one or more, i.e., at least one or two,according to the situation, 1–30, 1–20, or 1–10 amino acids ofN-terminal region of SEQ ID NO:1 or SEQ ID NO:2, further, or havingaddition of one or more, i.e., at least one or two, according to thesituation, 1–30, 1–20, or 1–10 amino acids to those N-terminus, withoutaltering the inherent activity. It is proper that the polypeptide of thepresent invention includes these mutants as far as they have desiredphysicochemical properties.

The polypeptide of the present invention can be obtained by the steps ofintroducing the DNA of the present invention into appropriate hosts, andcollecting from the culture of the transformants obtained. Thetransformant usable in the present invention is a transformantcontaining a DNA comprising, for example, nucleotide sequence, from the5′-terminus, of SEQ ID NO:3, SEQ ID NO:4, that having deletion,replacement or insertion of one or more nucleotides of those, anti-sensenucleotide sequence of those, or that having replacement of one or morenucleotides based on gene-degeneracy without altering the amino acidsequence encoded. The nucleotide sequence having replacement of one ormore, i.e., at least one or two, according to the situation, 1–190,1–60, or 1–30 nucleotides of SEQ ID:3 or SEQ ID NO:4 based ongene-degeneracy without altering the amino acid sequence encoded can beused as the nucleotide sequence described above.

The DNA of the present invention comprises a DNA originated from thenature and that synthesized artificially as far as the DNA has thenucleotide sequences described above. Microorganisms belonging the genusBacillus, for example, Bacillus globisporus C11 (FERM BP-7144) andBacillus globisporus N74 (FERM BP-7591) were usable as the naturalsources. A gene containing the DNA of the present invention can beobtained from the cells of these microorganisms. Specifically, a genecontaining the DNA can be released extracellularly by the steps ofinoculating the microorganism into a nutrient medium, culturing aboutone to three days under aerobic conditions, collecting the cells fromthe culture, treating the cells with cell-lysis enzymes such as lysozymeand β-glucanase or with ultrasonication. In addition to the methodsdescribed above, use of protein-hydrolyzing enzymes such as proteinases,detergents such as sodium dodecyl sulfate and freeze-thaw method arealso applicable. The objective DNA can be obtained from the disruptedcells using conventional methods in the art, for example, such asphenol-extraction, alcohol-precipitation, centrifugation andribonuclease-treatment. To synthesize the DNA artificially, chemicalsynthesis of the DNA based on the nucleotide sequence of SEQ ID NO:3 orSEQ ID NO:4 is applicable. PCR-method is also applicable to obtain theDNA using a gene containing the DNA as template and appropriatechemically synthetic DNA as a primer. The DNA can be obtained by thesteps of inserting the chemically synthetic DNA encoding SEQ ID NO:1 orSEQ ID NO:2 into appropriate autonomously replicable vector, introducingthe resultant recombinant DNA into appropriate hosts, culturing theresultant transformant, collecting the cells from the culture, andcollecting the recombinant DNA containing the DNA from the cells.

The DNAs are usually introduced into host-cells as the form ofrecombinant DNAs. Recombinant DNAs are usually constructed by a DNA andan autonomously replicable vector, and can be relatively easily preparedby the conventional recombinant DNA techniques if the DNA is obtained.The vectors, for instance, plasmid vectors such as pBR322, pUC18,Bluescript II SK(+), pUB110, pTZ4, pC194, pHV14, TRp7, YEp7 and pBS7; orphage vectors such as λgt·λc, λgt·λb, ρ11, φ1 and φ105 can be used. Toexpress the DNAs of the present invention in E. coli, pBR322, pUC18,Bluescript II SK(+), λgt·λc and λgt·λb are preferable. To express theDNAs of the present invention in B. subtilis, pUB110, pTZ4, pC194, ρ11,φ1 and φ105 are preferable. Plasmids, pHV14, TRp7, YEp7 and pBS7 areuseful in the case of replicating the recombinant DNAs in two or morehosts. In order to insert the DNA into these vectors, conventionalmethod used in the art can be used. Specifically, the DNA is insertedinto a vector by the steps of cleaving a gene containing the DNA andautonomously replicable vectors by restriction enzymes and/orultrasonication and ligating the resulting DNA fragment and theresulting vector fragment. The ligation of the DNA fragment and thevector fragment is easy by using a type II-restriction enzymes,particularly, such as Sau 3AI, Eco RI, Hind III, Ban HI, Sal I, Xba I,Sac I and Pst I, for cleaving genes and vectors. After the annealing ofthe both, if necessary, the desired recombinant DNA is obtainable byligating them in vivo or in vitro using a DNA ligase. The recombinantDNA, thus obtained, is unlimitedly replicable by the steps ofintroducing into appropriate hosts and culturing the resultanttransformants.

The recombinant DNA thus obtained can be introduced into appropriatehost-microorganisms such as E. coli, B. subtilis, Actinomyces andyeasts. The desired clones can be obtained from the transformants byapplying the colony-hybridization method or selecting by the steps ofculturing in nutrient media containing saccharides with a glucosepolymerization degree of 3 or higher and bearing both the α-1,6glucosidic linkage residue as a linkage at the non-reducing end and theα-1,4 glucosidic linkage other than the linkage at the non-reducing end,and selecting strains producing cyclotetrasaccharide from thesaccharides.

The transformants, thus obtained, produce the polypeptide of the presentinvention extra/intracellularly when cultured in nutrient media.Conventional liquid media which are supplimented with carbon sources,nitrogen sources and minerals, furthermore, if necessary, withtrace-nutrients such as amino acid and vitamins, are usually used as thenutrient media. Examples of carbon sources are saccharides includingstarch, starch hydrolyzate, glucose, fructose, sucrose, α,α-trehalose,α,β-trehalose and β,β-trehalose. Examples of nitrogen sources arenitrogen-containing inorganic- or organic-substances including ammonia,ammonium salts, urea, nitrate, peptone, yeast extract, defatted soybean,corn-steep liquor and meat extract. Cultures containing the polypeptideare obtainable by the steps of inoculating the transformants into thenutrient media, culturing for about one to six days under aerobicconditions such as aeration and agitation conditions while keeping thetemperature and pH, usually, at 20–40° C., and pH 2–10. Although theculture can be used intact for enzyme preparation, the polypeptides ofthe present invention are usually, if necessary, separated from cells orcell debris and purified before use by filtration or centrifugationafter extracting from cells using osmotic shock procedure ordetergent-treatment, or disrupting cells by ultrasonication or usingcell-lysis enzymes. The polypeptides can be purified by applying thepurification procedures for polypeptide commonly used, for example,appropriate combination of one or more procedures such as concentration,salting out, dialysis, precipitation, gel filtration chromatography,ion-exchange chromatography, hydrophobic chromatography, affinitychromatography, gel electrophoresis and isoelectrofocusing.

The polypeptides of the present invention have unique properties offorming a cyclotetrasaccharide from saccharides with a glucosepolymerization degree of 3 or higher and bearing both theα-1,6-glucosidic linkage as a linkage at the non-reducing end and theα-1,4 glucosidic linkage other than the linkage at the non-reducing end,and comprising amino acid sequences of SEQ ID NO:1, SEQ ID NO:2 or theamino acid sequences having deletion, replacement or insertion of one ormore amino acids of SEQ ID NO:1 or SEQ ID NO:2. Cyclotetrasaccharideproduced by the action of the polypeptide of the present invention showsno amino-carbonyl reactivity and less browning and deterioration becauseof its non-reducibility. The saccharide also has an inclusion ability ofvolatile substances such as ethyl alcohol and acetic acid because of itscyclic structure. Furthermore, cyclotetrasaccharide has useful featuressuch as mild and low sweetness, which less spoil the inherent tastes offoods by excessive sweetness, low-fermentability and low digestibilitygood for dietary-fibers.

The following explains the formation of cyclotetrasaccharide.Cyclotetrasaccharide can be obtained by acting the polypeptide of thepresent invention on the substrates, saccharides with a glucosepolymerization degree of 3 or higher and bearing both the α-1,6glucosidic linkage as a linkage at the non-reducing end and α-1,4glucosidic linkage other than the linkage at the non-reducing end. Thesaccharides can be obtained as transfer-products by actingα-glucosidase, dextrindextranase or α-isomaltosylglucosaccharide-formingenzyme which is disclosed in PTC/JP01/06412 by the present inventors onstarch, starchy compounds such as amylopectin, amylose and glycogen, orthose partial hydrolyzates obtained by using acids and/or amylases. Thesaccharides can also be obtained by acting β-amylase and pullulanase onpullulan. Examples of these saccharide are one or more saccharides witha glucose polymerization degree of 3 or higher and bearing both theα-1,6 glucosidic linkage as a linkage at the non-reducing end and α-1,4glucosidic linkage other than the linkage at the non-reducing end suchas 6²-O-α-glucosylmaltose, 6³-O-β-glucosylmaltotriose,6⁴-O-α-glucosylmaltotetraose and 6⁵-O-α-glucosylmaltopentaose.

In the process for the production of cyclotetrasaccharide, thepolypeptide of the present invention can be advantageously added to actin the beginning, course, or end of the formation of the saccharide witha glucose polymerization degree of 3 or higher and bearing both theα-1,6 glucosidic linkage as a linkage at the non-reducing end and theα-1,4 glucosidic linkage other than the linkage at the non-reducing end.Usually, the polypeptide of the present invention is allowed to act onappropriate solutions containing one or more saccharides described aboveas the substrate with keeping desired temperature and pH until whendesired amount of cyclotetrasaccharide is formed. Although the enzymaticreaction proceeds under the substrate concentration of about 0.1% (w/w),one percent or higher substrate concentration (throughout thespecification, “%(w/w)” is abbreviated as “%” hereinafter, unlessspecified otherwise), more preferably, 5–50% is used for an industrialscale production. The temperatures for the enzymatic reaction used inthe present invention are those which proceed the enzymatic reaction,i.e., those up to about 60° C., preferably, about 30° C. to about 50° C.The pHs for the enzymatic reaction are usually set to 4.5 to 8,preferably about 5.5 to about 7. Since the amount of the polypeptide ofthe present invention is closely related to the time for the reaction,those can be appropriately set depending on the enzymatic reactionefficiency. The polypeptide can be advantageously used as an immobilizedpolypeptide by immobilizing it to appropriate carriers usingconventional procedures.

The reaction mixture, obtained from the reaction described above,usually includes cyclotetrasaccharide, glucose, maltodextrins such asmaltose, a saccharide having a glucose polymerization degree of 3 orhigher and having both the α-1,6 glucosidic linkage as a linkage at thenon-reducing end and the α-1,4 glucosidic linkage other than the linkageat the non-reducing end, and can be used intact ascyclotetrasaccharide-containing solution. After allowing the polypeptideof the present invention to act the substrate, if necessary,contaminating oligosaccharides in the solution can be hydrolyzed by oneor more enzymes selected from the group comprising α-amylase, β-amylase,glucoamylae, and α-glucosidase. Usually, the sugar solution can be usedafter further purification. One or more conventional methods, forexample, selected from the group of decolorization with activatedcharcoal, desalting by H— or OH—form ion exchanger resin, and columnchromatographies such as ion-exchange column chromatography, activatedcharcoal column chromatography, and silica gel column chromatography,separation using organic solvents such as alcohol and acetone, membraneseparation using adequate separability, fermentation by microorganismcapable of utilizing or decomposing the contaminating saccharideswithout utilizing cyclotetrasaccharide, such as Lactobacillus,Acetobacter and yeast, and alkaline-treatment to decompose the remainingreducing sugars can be advantageously used as the purificationprocedures. Particularly, ion-exchange chromatography is preferably usedas an industrial scale production method; column chromatography usingstrong-acid cation exchange resin as disclosed, for example, in JapanesePatent Kokai Nos. 23,799/83 and 72,598/98. Using the columnchromatography, the contaminating saccharide can be removed toadvantageously produce cyclotetrasaccharide with an improved content ofthe objective saccharide or saccharide compositions comprising the same.In this case, any one of fixed-bed, moving bed, semi-moving bed, batch,semi-continuous, and continuous methods can be appropriately used.

The resulting cyclotetrasaccharide or saccharide compositions comprisingthe same with an improved content are aqueous solutions containingcyclotetrasaccharide, usually 10% or more, d.s.b., preferably 40% ormore, d.s.b. Usually, the resulting cyclotetrasaccharide or saccharidecompositions comprising the same can be concentrated into syrupproducts, and optionally they can be further dried into powderyproducts. To produce cyclotetrasaccharide crystals, usually saccharidesolution comprising cyclotetrasaccharide purified as described above,preferably cyclotetrasaccharide solution, having a concentration ofabout 40% or more, d.s.b., can be used. In the case to producecyclotetrasaccharide penta- to hexa-hydrate crystals, usually, thesaccharide solutions are brought to supersaturated solution, forexample, having a concentration of about 40–90%, and are placed in acrystallizer, and then gradually cooled while stirring in the presenceof 0.1–20%, d.s.b., of a seed crystal with a temperature keepingsuper-saturation, preferably, 10–90° C., to produce massecuitescontaining the crystals. In the case to produce cyclotetrasaccharidemono-hydrate or anhydrous crystals, the super-saturation conditions ofhigher temperature and higher concentration are used. The methods tocollect cyclotetrasaccharide crystals and molasses with such crystalsinclude, for example, conventional methods such as separation, blockpulverization, fluidized granulation, and spray drying methods.Cyclotetrasaccharide mono-hydrate and anhydrous crystals can be producedby dehydrating and drying cyclotetrasaccharide penta- to hexa-hydratecrystals. The resulting cyclotetrasaccharide crystal or highcyclotetrasaccharide content powder is non-reducing or less reducingwhite powder having delicate and mild low-sweetness, and is stablesaccharide having high tolerance to acid and thermal stability. Thepowder is almost free of browning, smelling and deterioration ofmaterials even when mixed or processed therewith: the materials areparticularly, for example, amino acid-containing substances such asamino acids, oligopeptides, and proteins. Furthermore, the powder haslow hygroscopicity and is capable of preventing adhesion andsolidification of powdery substances.

Since cyclotetrasaccharide has inclusion ability, it effectivelyinhibits the dispersion and quality deterioration of flavorfulcomponents and effective ingredients. Therefore, cyclotetrasaccharidecan be advantageously used as flavor-retaining agent and stabilizer. Forsuch a purpose, if necessary, the combination use ofcyclotetrasaccharide and other cyclic sacchride(s) such ascyclodextrins, branched cyclodextrins, cyclodextrans and cyclofructanscan be advantageously used to improve the stabilizing effects.

Since cyclotetrasaccharide is not hydrolyzed by amylase andα-glucosidase, it is substantially free of assimilation by the body whenorally administrated. Also, the saccharide is not substantiallyassimilated by intestinal bacteria, and therefore it can be used as anextremely-low caloric water-soluble dietary fiber. In other words,although cyclotetrasaccharide has a weight and volume to give a feelingof fullness, it is not substantially assimilated when orallyadministrated. Therefore, it can be advantageously used as low-caloricfood material and dietary food material. Cycoltetrasaccharide can bealso used as a sweetener substantially free from causing dental cariesbecause it is scarcely assimilated by dental caries-inducing bacteria.

Cyclotetrasaccharide per se is a natural sweetener with a goodacid-tolerance, alkaline-tolerance and thermal stability but with notoxicity and harm. Because of these, in the case of crystalline product,it can be advantageously used for tablets and sugar-coated tablets incombination with binders such as pullulan, hydroxyethyl starch, andpolyvinylpyrrolidone. Furthermore, cyclotetrasaccharide has propertiesof osmosis-controlling ability, filler-imparting ability,gloss-imparting ability, moisture-retaining ability, viscosity,syneresis -preventing ability, solidification-preventing ability,flavor-retaining ability, stability, crystallization-preventing abilityfor other sugars, insubstantial fermentability, starchretrogradation-preventing ability, protein denaturation-preventingability, lipid deterioration-preventing ability, etc.

Thus, cyclotetrasaccharide and the saccharide compositions comprisingthe same can be used intact as a sweetener, low-fermentable foodmaterial, low-digestive food material, low-cariogenic food material,low-caloric food material, taste-improving agent, flavor-improvingagent, quality-improving agent, preventive of syneresis, preventive ofsolidification, flavor-retaining agent, preventive of starchretrogradation, preventive of protein denaturation, preventive of lipiddeterioration, stabilizer, excipient, inclusion agent, base ofpulverization, etc. If necessary, the combination use ofcyclotetrasaccharide and conventional materials can be advantageouslyused as various compositions, for example, food products, tobacco,cigarette, feeds, pet foods, cosmetics, and pharmaceuticals. seasoning,color-imparting agent, flavor-imparting agent, reinforcing agent,emulsifying agent, preventive of oxidation, preventive of ultravioletrays, and efficacy components of medicine can be appropriately used asthe conventional materials.

Cyclotetrasaccharide and the saccharide compositions comprising the samecan be used intact as sweeteners. If necessary, they can beadvantageously used in combination with other sweeteners, for example,powdery syrup, glucose, fructose, isomerized sugar, sucrosd, maltose,α,α-trehalose, α,β-trehalose, β,β-trehalose, honey, maple sugar,erythritol, xylitol, sorbitol, maltitol, deihydrochalcone, stevioside,α-glycosyl stevioside, sweetener of Momordica grosvenori, glycyrrhizin,thaumatin, L-aspartyl L-phenylalanine methyl ester, saccharine,acesulfame K, sucralose, glycine and alanine; and fillers such asdextrin, starch, and lactose. Particularly, cyclotetrasaccharide and thesaccharide compositions comprising the same can be suitably used as alow caloric sweetener, dietary sweetener, or the like in combinationwith one or more low-caloric sweeteners such as erythritol, xylitol, andmaltitol; and/or one or more sweeteners with a relatively-highsweetening power such as α-glycosyl stevioside, thaumatin, L-aspartylL-phenylalanine methyl ester, saccharine, acesulfame K, and sucralose.

Powdery and/or crystalline products of cyclotetrasaccharide and thesaccharide compositions comprising the same can be arbitrarily usedintact or, if necessary, after mixing with fillers, excipients, binders,etc., and them formed into products with different shapes such asgranules, spheres, sticks, plates, cubes, and tablets.

Cyclotetrasaccharide and the saccharide compositions comprising the samewell harmonize with other tastable materials having sour-, salty-,bitter-, astringent-, delicious, and bitter-taste; and have a high acid-and heat-tolerance. Thus, they can be favorably used as sweeteners,taste-improving agent, flavor-improving agent, quality-improving agent,etc., to sweeten and/or improve the taste, flavor, and quality of foodproducts in general, for example, a soy sauce, powdered soy sauce, miso,“funmatsu-miso” (a powdered miso), “moromi” (a refined sake), “hishlo”(arefined soy sauce), “furikake” (a seasoned fish meal), mayonnaise,dressing, vinegar, “sanbai-zu” (a sauce of sugar, soy sauce andvinegar), “funmatsu-sushi-zu” (powdered vinegar for sushi),“chuka-no-moto” (an instant mix for Chinese dish), “tentsuyu” (a saucefor Japanese deep fat fried food), “mentsuyu” (a sauce for Japanesevermicelli), sauce, catsup, “yakiniku-no-tare” (a sauce for Japanesegrilled meat), curry roux, instant stew mix, instant soup mix,“dashi-no-moto” (an instant stock mix), mixed seasoning, “mirin” (asweet sake), “shin-mirin” (a synthetic mirin), table sugar, and coffeesugar. Also, cyclotetrasaccharide and the saccharide compositionscomprising the same can be arbitrarily used to sweeten and improve thetaste, flavor, and quality of “wagashi” (Japanese cakes) such as“senbei” (a rice cracker), “arare” (a rice cake cube), “okoshi” (amillet and rice cake), “gyuhi” (a starch paste), “mochi” (a rise paste)and the like, “manju” (a bun with a bean-jam), “uiro” (a sweet ricejelly), “an” (a bean-jam) and the like, “yokan” (a sweet jelly ofbeans), “mizu-yokan” (a soft azuki-bean jelly), “kingyoku” (a kind ofyokan), jelly, pao de Castella, and “amedama” (a Japanese toffee);Western confectioneries such as a bun, biscuit, cracker, cookie, pie,pudding, butter cream, custard cream, cream puff, waffle, sponge cake,doughnut, chocolate, chewing gum, caramel, nougat, and candy; frozendesserts such as an ice cream and sherbet; syrups such as a“kajitsu-no-syrup-zuke” (a preserved fruit) and “korimitsu” (a sugarsyrup for shaved ice); pastes such as a flour paste, peanut paste, andfruit paste; processed fruits and vegetables such as a jam, marmalade,“syrup-zuke” (fruit pickles), and “toka” (conserves); pickles andpickled products such as a “fukujin-zuke” (red colored radish pickles),“bettara-zuke” (a kind of whole fresh radish pickles), “senmai-zuke” (akind of sliced fresh radish pickles), and “rakkyo-zuke” (pickledshallots); premix for pickles and pickled products such as a“takuan-zuke-no-moto” (a premix for pickled radish), and“hakusai-zuke-no-moto” (a premix for fresh white rape pickles); meatproducts such as a ham and sausage; products of fish meat such as a fishham, fish sausage, “kamaboko” (a steamed fish paste), “chikuwa” (a kindof fish paste), and “tenpura” (a Japanese deep-fat fried fish paste);“chinmi” (relish) such as a “uni-no-shiokara” (salted guts of urchin),“ika-no-shiokara” (salted guts of squid), “su-konbu” (processed tangle),“saki-surume” (dried squid strips), “fugu-no-mirin-boshi” (a driedmirin-seasoned shellfish), seasoned fish flour such as of Pacific cod,sea bream, shrimp, etc.; “tsukudani” (foods boiled down in soy sauce)such as those of laver, edible wild plants, dried squid, small fish, andshellfish; daily dishes such as a “nimame” (cooked beans), potato salad,and “konbu-maki” (a tangle roll); milk products; canned and bottledproducts such as those of meat, fish meat, fruit, and vegetable;alcoholic beverages such as a synthetic sake, fermented liquor, fruitliquor, and sake; soft drinks such as a coffee, cocoa, juice, carbonatedbeverage, sour milk beverage, and beverage containing a lactic acidbacterium; instant food products such as instant pudding mix, instanthot cake mix, instant juice, instant coffee, “sokuseki-shiruko” (aninstant mix of azuki-bean soup with rice cake), and instant soup mix;and other foods and beverages such as solid foods for babies, foods fortherapy, drinks, beverage containing amino acids, peptide foods, andfrozen foods.

Cyclotetrasaccharide and the saccharide compositions comprising the samecan be arbitrarily used to improve the taste preference or to reduce thecalorie of feeds and pet foods for animals and pets such as domesticanimals, poultry, honey bees, silk worms, and fish; and also they can bearbitrarily used as a sweetener and taste-improving agent, taste-curingagent, quality-improving agent, and stabilizer in other products in apaste or liquid form such as tobacco, cigarette, tooth paste, lipstick,rouge, lip cream, internal liquid medicine, tablet, troche, cod-liveroil in the form of drop, oral refrigerant, cachou, gargle, cosmetic andpharmaceutical. When used as a quality-improving agent or stabilizer,cyclotetrasaccharide and the saccharide compositions comprising the samecan be arbitrarily used in biologically active substances susceptible tolose their effective ingredients and activities, as well as in healthfoods, cosmetics, and pharmaceuticals containing the biologically activesubstances. Example of such biologically active substances are liquidpreparations containing cytokines such as α-, β-, and γ-interferons,tumor necrosis factor-α (TNF-α), tumor necrosis factor-β (TNF-β),macropharge migration inhibitory factor, colony-stimulating factor,transfer factor, and interleukin 2; liquid preparations containinghormones such as insulin, growth hormone, prolactin, erythropoietin, andfollicle-stimulating hormone; biological preparations such as BCGvaccine, Japanese encephalitis vaccine, measles vaccine, live poliovaccine, small pox vaccine, tetanus toxoid, Trimeresurus antitoxin, andhuman immunoglobulin; antibiotics such as penicillin, erythromycin,chloramphenicol, tetracycline, streptmycin, and kanamycin sulfate;vitamins such as thiamin, ribofravin, L-ascorbic acid, cod liver oil,carotenoide, ergosterol, tocopherol; solution of enzymes such as lipase,esterase, urokinase, protease, β-amylase, isoamylase, glucanase, andlactase; extracts such as ginseng extract, turtle extract, chlorellaextract, aloe extract, bamboo-leaf extract, peach-leaf extract,loquat-leaf extract, citron-peel extract, and propolis extract;biologically active substances such as living microorganisms paste ofvirus, lactic acid bacteria, and yeast, and royal jelly. By usingcyclotetrasaccharide and the saccharide compositions comprising thesame, the above biologically active substances can be arbitrary preparedin health foods, cosmetics, and pharmaceuticals in a liquid, paste, orsolid form, which have a satisfactorily-high stability and quality withless fear of losing or inactivating their effective ingredients andactivities.

The methods for incorporating cyclotetrasaccharide or the saccharidecomposition comprising the same into the aforesaid compositions arethose which can incorporate cyclotetrasaccharide and the saccharidecompositions into a variety of compositions before completion of theirprocessing, and which can be appropriately selected from the followingconventional methods; mixting, kneading, dissolving, melting, soaking,penetrating, dispersing, applying, coating, spraying, injecting,crystallizing, and solidifying. In order to exercise the variouscharacteristics of cyclotetrasaccharide, particularly, inclusionability, taste-improving ability, and flavor-improving ability, theamount of cyclotetrasaccharide or the saccharide compositions comprisingthe same to be preferably incorporated into the final compositions isusually in an amount of 0.1% or more, desirably, 1% or more.

The following examples explain in detail the production processes forthe polypeptide of the present invention, cyclotetrasaccharideobtainable thereby, and saccharides comprising the same:

EXAMPLE 1

Production of a Polypeptide

A liquid medium containing 5 g/L of “PINE-DEX #4”, a partial starchhydrolyzate, 20 g/L of polypeptone, 20 g/L of yeast extract, 1 g/L ofsodium phosphate, and water was placed in a 500-ml Erlenmeyer flask inan amount of 100 ml, sterilized at 121° C. for 15 min, and cooled. Then,the liquid medium was sterilely set to pH 7.0, and admixed withampicillin sodium salt to give a final concentration of 100 μg/ml. Atransformant, BGC1, obtained by the method in Experiment 5-2, wasinoculated into the above liquid medium, and cultured at 27° C. and at230 rpm for 24 hours to obtain the seed culture. Subsequently, about 18L of a fresh preparation of the same liquid culture medium as used aboveseed culture was placed in a 30-L fermentor, sterilized with the samemanner, cooled to 27° C., and then admixed with ampicillin to give aconcentration of 50 μg/ml, and inoculated with 1%(v/v) of the seedculture, followed by culturing at 27° C. for 48 hours underaeration-agitation conditions. After disrupting cells in the culture byultrasonication and removing the cell-debris by centrifugation, theactivity of the polypeptide of the present invention in the resultingsupernatant was assayed. The supernatant had about 3,100 units/L ofα-isomaltosyl-transferring enzyme activity. About 74 ml of enzymesolution containing about 135 units/ml of the polypeptide of the presentinvention, having α-isomaltosyl-transferring enzyme activity, whosespecific activity is about 30 units/mg-protein, was obtained bypurifying the supernatant according to the method described inExperiment 1.

EXAMPLE 2

Production of a Polypeptide

According to the method described in Example 1, BGN1, a transformantobtained in Experiment 6-2, was seed-cultured, and then main-culturedusing a 30-L fermentor. After disrupting cells in the culture byultrasonication and removing the cell-debris by centrifugation, theactivity of the polypeptide of the present invention in the resultingsupernatant was assayed. The supernatant had about 3,000 units/L ofα-isomaltosyl-transferring enzyme activity. About 150 ml of enzymesolution containing about 72 units/ml of the polypeptide of the presentinvention, having α-isomaltosyl-transferring enzyme activity, whosespecific activity is about 30 units/mg-protein, was obtained bypurifying the supernatant according to the method described inExperiment 3.

EXAMPLE 3

Production of a Powdery Product Containing Cyclotetrasaccharide

To an aqueous solution containing 10% panose, commercialized byHayashibara Biochemical Laboratories Inc., set at pH 6.0 and 35° C.,enzyme polypeptide obtained by the method described in Example 1 wasadded to give a concentration 2 units/g-panose and incubated for 36hours. The reaction mixture was heated to 95° C. and kept for 10 minute,and then cooled and filtered to obtain a filtrate. According to theconventional manner, the resulting filtrate was decolored with activatedcharcoal, desalted and purified with ion exchangers in H- and OH- forms,and then concentrated and dried into a powdery products containingcyclotetrasaccharide in a yield of about 91%, d.s.b.

Since the product contains, on a dry solid basis, 34.0% glucose, 2.1%isomaltose, 2.3% panose, 45.0% cyclotetrasaccharide, 4.8%isomaltosylpanose, 1.8% isomaltosylpanoside, and 10.0% of othersaccharides and has a mild sweetness, an adequate viscosity,moisture-retaining ability, and inclusion ability, it can beadvantageously used in a variety of compositions such as food products,cosmetics, and pharmaceuticals as a sweetener, taste-improving agent,quality-improving agent, syneresis-preventing agent, stabilizer,excipient, inclusion agent, and base of pulverization.

EXAMPLE 4

Production of a Syrupy Composition Containing Cyclotetrasaccharide

“SUNMALT®”, a powdery maltose commercialized by Hayashibara Co., Ltd.,was dissolved into water to give a concentration of 30% and admixed with0.08%, d.s.b., of “TRANSGLUCOSIDASE L AMANO™”, an α-glucosidasecommercialized by Amano Pharmaceutical Co., Ltd., and then set to pH5.5, followed by the enzymatic reaction at 55° C. for 18 hours. Afterstopping the reaction by heating, the reaction mixture was set to pH 6.0and 35° C., and admixed 2 units/g-dry solid basis of enzyme polypeptideobtained in Example 1, and then incubated for 36 hours. The reactionmixture was heated to 95° C. and kept for 10 minute, and then cooled andfiltered to obtain a filtrate. According to the conventional manner, theresulting filtrate was decolored with activated charcoal, desalted andpurified with ion exchangers in H— and OH— forms, and then concentratedinto a 70% syrup in a yield of about 92%, d.s.b.

Since the product contains, on a dry solid basis, 32.5% glucose, 15.7%maltose, 9.8% isomaltose, 4.0% maltotriose, 0.3% panose, 1.6%isomaltotriose, 17.5% cyclotetrasaccharide, 1.2% isomaltosylpanose, 0.7%isomaltosylpanoside, and 16.7% of other saccharides and has a mildsweetness, an adequate viscosity, moisture-retaining ability, andinclusion ability, it can be advantageously used in a variety ofcompositions such as food products, cosmetics, and pharmaceuticals as asweetener, taste-improving agent, quality-improving. agent,syneresis-preventing agent, stabilizer, excipient, inclusion agent, andbase of pulverization.

EXAMPLE 5

Production of a Crystalline Powder of Cyclotetrasaccharide

A potato starch was prepared into a 15% starch suspension, admixed withcalcium carbonate to give a final concentration of 0.1%, adjusted to pH6.0, and admixed with 0.2%/g-starch of “THERMAMYL 60 L”, an α-amylasecommercialized by Novo Industries A/S, Copenhagen, Denmark, and thenheated at 95° C. for 15 min. After autoclaving at 2 kg/cm2 for 30 min,the reaction mixture was cooled to 35° C., admixed with 7.5units/g-starch of the polypeptide of the present invention, obtained inExample 1, 2 units/g-starch of α-isomaltosylglucosaccharide-formingenzyme obtained by the method in Experiment 1-3, and 10 units/g-starchof cyclomaltodextrin glucanotransferase commercialized by HayashibaraBiochemical Laboratories Inc., followed by the enzymatic reaction for 48hours. After heating to 95° C. for 30 min, the reaction mixture wasadjusted at 5%, pH 5.0, and 45° C., admixed with 1,500 units/g-starch of“TRANSGLUCOSIDASE L AMANO™”, an α-glucosidase and 75 units/g-starch of“GLUCOZYME”, a glucoamylase preparation commercialized by NagaseBiochemicals, Ltd, Kyoto, Japan, and then enzymatically reacted for 24hours. The reaction mixture was heated to 95° C. and kept for 10 minute,and then cooled and filtered to obtain a filtrate. According to theconventional manner, the resulting filtrate was decolored with activatedcharcoal, desalted and purified with ion exchangers in H— and OH— forms,and then concentrated into a 60% syrup. The resulting syrup contained,on a dry solid basis, 27.5% glucose, 65.1% cyclotetrasaccharide, and7.5% of other saccharides. The resulting saccharide solution wassubjected to a column chromatography using “AMBERLITE CR-1310(Na-form)”, a strong acid cation-exchanger resin commercialized by JapanOrgano Co., Ltd., Tokyo, Japan. The resin was packed into four jacketedstainless steel columns having a diameter of 5.4 cm, which were thencascaded in series to give a total gel bed depth of 20 m. Under theconditions of keeping the inner column temperature at 60° C., thesaccharide solution was fed to the columns in a volume of 5%(v/v) andfractionated by feeding to the columns hot water heated to 60° C. at anSV (space velocity) of 0.13 to obtain high cyclotetrasaccharide contentfractions while monitoring the saccharide composition of eluate by HPLC,and then collected the high cyclotetrasaccharide content fractions. Thehigh cyclotetrasaccharide content solution was obtained in a yield ofabout 21%, d.s.b. The solution contained about 98%, d.s.b. ofcyclotetrasaccharide.

The solution was concentrated to give a concentration of about 70% andthen placed in a crystallizer, admixed with about 2% crystallinecyclotetrasaccharide penta- or hexa-hydrate as seed crystal, andgradually cooled to obtain a massecuite with a crystallinity of about45%. The massecuite was sprayed from a nozzle equipped on top of dryingtower at high pressure of 150 kg/cm². Simultaneously, hot air heated to85° C. was drawn down from the upper part of the drying tower, and theresulting crystal powder was collected on a transporting wire conveyorprovided on the basement of the tower and gradually moved out of thetower while blowing thereunto a hot air heated to 45° C. The resultingcrystalline powder was injected to an ageing tower and aged for 10 hourswhile a hot air was being blown to the contents to completecrystallization and drying to obtain a crystalline powder ofcyclotetrasaccharide penta- or hexa-hydrate.

Since the product has a relatively low reducibility, does substantiallyneither cause the amino-carbonyl reaction nor exhibit hygroscopicity,and has a satisfactory handleability, mild low sweetness, adequateviscosity, moisture-retaining ability, inclusion ability, andsubstantially non-digestibility, it can be advantageously used in avariety of compositions such as food products, cosmetics, andpharmaceuticals as a sweetener, low calorie food, taste-improving agent,flavor-improving agent, quality-improving agent, syneresis-preventingagent, stabilizer, excipient, inclusion agent, and base ofpulverization.

EXAMPLE 6

Production of a Crystalline Powder of Cyclotetrasaccharide

A corn starch was prepared into a 28% starch suspension, admixed withcalcium carbonate to give a concentration of 0.1%, adjusted to pH 6.5,and admixed with 0. 3%/g-starch of “THERMAMYL 60 L”, an α-amylasecommercialized by Novo Industries A/S, Copenhagen, Denmark, and thenheated at 95° C. for 15 min. After autoclaving at 2 kg/cm² for 30 min,the reaction mixture was cooled to 50° C., admixed with 6 units/g-starchof the polypeptide of the present invention, obtained in Example 2, 1.8units/g-starch of α-isomaltosylglucosaccharide-forming enzyme obtainedby the method in Experiment 3-3, and one units/g-starch ofcyclomaltodextrin glucanotransferase commercialized by HayashibaraBiochemical Laboratories Inc., followed by the enzymatic reaction for 72hours. After heating to 95° C. for 30 min, the reaction mixture wasadjusted to pH 5.0, and 50° C., admixed with 300 units/g-starch of“TRANSGLUCOSIDASE L AMANO™”, an α-glucosidase, reacted for 24 hours, andthen admixed with 10 units/g-d.s.b., of “GLUCOZYME”, a glucoamylasepreparation commercialized by Nagase Biochemicals, Ltd, Kyoto, Japan,and 20 units/g-d.s.b., of “NEO-SPITASE PK2”, an α-amylase preparation,and then reacted for 17 hours. The reaction mixture was heated to 95° C.and kept for 30 minute, and then cooled and filtered to obtain afiltrate. According to the conventional manner, the resulting filtratewas decolored with activated charcoal, desalted and purified with ionexchangers in H— and OH— forms, and then concentrated into a 60% syrup.The resulting syrup contained, on a dry solid basis, 35.1% glucose,51.1% cyclotetrasaccharide, and 13.8% of other saccharides. Theresulting saccharide solution was fractionated by a columnchromatography using a strong acid cation-exchanger resin described inExample 5, and then collected the high cyclotetrasaccharide contentfractions in a yield of about 39%, d.s.b. The solution contained about80%, d.s.b., of cyclotetrasaccharide.

The solution was continuously crystallized while concentrating. Theresulting massecuite was separated by a basket-type centrifuge to obtaincrystals which were then sprayed with a small amount of water to obtaina high purity cyclotetrasaccharide, penta- or hexa-hydrate, in a yieldof about 23%, d.s.b.

Since the product has a relatively low reducibility, does substantiallyneither cause the amino-carbonyl reaction nor exhibit higroscopicity,and has a satisfactory handleability, mild low sweetness, adequateviscosity, moisture-retaining ability, inclusion ability, andsubstantially non-digestibility, it can be advantageously used in avariety of compositions such as food products, cosmetics, andpharmaceuticals as a sweetener, low calorie food, taste-improving agent,flavor-improving agent, quality-improving agent, syneresis-preventingagent, stabilizer, excipient, inclusion agent, and base ofpulverization.

INDUSTRIAL APPLICABILITY

As described above, the present invention is an invention providing anovel polypeptide which have α-isomaltosyl transferring activity, andits process and uses. The polypeptide of the present invention can bestably provided in large amount and at a relatively low cost byrecombinant DNA techniques. Therefore, According to the presentinvention, a cyclotetrasaccharide having a structure ofcyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→},saccharide mixture comprising the same, and a variety of compositionscomprising the same can be stably produced in an industrial scale and ata relatively low cost. Since the cyclotetrasacchride has are lativelylow reducibility, does substantially neither cause the amino-carbonylreaction nor exhibit higroscopicity, and has a satisfactoryhandleability, mild low sweetness, adequate viscosity,moisture-retaining ability, inclusion ability, and substantiallynon-digestibility, it can be advantageously used in a variety ofcompositions such as food products, cosmetics, and pharmaceuticals as asweetener, low calorie food, taste-improving agent, flavor-improvingagent, quality-improving agent, syneresis-preventing agent, stabilizer,excipient, inclusion agent, and base of pulverization.

The present invention, having these outstanding functions and effects,is a significantly important invention that greatly contributes to thisart.

1. An isolated DNA, which encodes a polypeptide comprising the aminoacid sequence of SEQ ID NO:2; wherein the polypeptide has an enzymaticactivity of producing, through α-isomaltosyl-transferring reaction, asaccharide having a structure of cyclo{ 6)-α-D-glucopyranosyl-(13)-α-D-glucopyranosyl-(1 6)-α-D-glucopyranosyl-(13)-α-D-glucopyranosyl-(1 } from a saccharide having a glucosepolymerization degree of 3 or higher and having both α-1,6 glucosidiclinkage as a linkage at the non-reducing end and α-1,4 glucosidiclinkage other than the linkage at the non-reducing end, represented bythe chemical formula 1:6^(n)-O-α-glucosyl-Gn  Chemical formula 1 (wherein “Gn” means anα-1,4-glucan having a glucose polymerization degree of “n”, and “n”means an integer of 2 or greater.).
 2. The isolated DNA of claim 1,which comprises the nucleotide sequence of SEQ ID NO:4; or thenucleotide sequence having replacement of one or more nucleotides of SEQID NO:4 without changing the amino acid sequence of SEQ ID NO:2 based ongenetic code degeneracy, or fully complementary nucleotide sequencesthereof.
 3. The isolated DNA of claim 1, which originates from amicroorganism of the genus Bacillus.
 4. A replicable recombinant DNA,which comprises the DNA of claim 1 and an autonomously replicablevector.
 5. The replicable recombinant DNA of claim 4, wherein saidautonomously-replicable vector is a plasmid vector, Bluescript II SK(+).6. An isolated transformed cell, which is constructed by introducing therecombinant DNA of claim 4 into an appropriate host-microorganismselected from the group consisting of Escherichia coli, Bacillussubtilis, Actinomyces and yeasts.
 7. The tranaformant isolatedtransformed cell of claim 6, wherein said host is Escherichia coli.
 8. Aprocess for producing a polypeptide comprising the amino acid sequenceof SEQ ID NO: 2 comprising the steps of culturing the isolatedtransformed cell of claim 6 and collecting the polypeptide from theresulting cell culture.
 9. The process of claim 8, wherein thepolypeptide is collected by one or more techniques selected from thegroup consisting of centrifugation, filtration, concentration, saltingout, dialysis, concentration, separatory precipitation, ion-exchangechromatography, gel filtration chromatography, hydrophobicchromatography, affinity chromatography, gel electrophoresis, andisoelectric focusing.